Synthesis and antimicrobial activity of certain 6H-1,2,4-oxadiazin-3(2H)-ones. Treatment of 6H-1,2,4-oxadiazin-3(2H)-one-5(4H)-thione (2) with hydroxylamine, hydrazine, methylamine, or benzylamine afforded the corresponding N5-substituted 5-amino-6H-1,2,4-oxadiazin-3(2H)-ones 3c-f. Refluxing a dioxane solution of 6H-1,2,4-oxiazine-3,5(2H,4H)-dione (1) with benzylamine or aminodiphenylmethane and hexamethyldisilazane in the presence of ammonium sulfate gave 5-benzylamino-6H-1,2,4-oxadiazin-3(2H)-one (3f) and the corresponding 5-diphenylmethylamino derivative 3g. Reaction of 1 with methyl iodide, benzyl chloride, dihydropyran, dihydrofuran, or benzyloxycarbonyl chloride afforded the corresponding 2-substituted 6H-1,2,4-oxadiazine-3,5(2H,4H)-diones 6a-e. Reaction of 2-methyl-6H-1,2,4-oxadiazine-3,5(2H,4H)-dione (6a) or the corresponding 2-benzyl derivative 6b with phosphorus pentasulfide in dioxane gave 2-methyl-6H-1,2,4-oxadiazin-3(2H)-one-5(4H)-thione (8a) and the corresponding 2-benzyl derivative 8b, respectively. Reaction of 8a with ammonia in dioxane afforded 2-methyl-5-amino-6H-1,2,4-oxadiazin-3(2H)-one (9). The degree of in vitro activity and the presence of antibacterial activity in the urine of animals given 5-amino-6H-1,2,4-oxadiazin-3(2H)-one (3a) by oral route of administration prompted selection of this compound for further study.


Mitomycin antibiotics. Synthesis and activity of 1,2-disubstituted mitosenes. cis-1-Acetamido-2-acetoxy-7-methoxy-N-methylmitosene was prepared in 11 steps from 7-methoxy-6-methyl-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-one by a route involving bromination of the pyrrolidineenamine or trimethylsilyl enol ether of starting material, displacement of bromide by acetate, oxime formation, and reductive acetylation, followed by elaboration of the quinone and methyl carbamate functions according to previously established methods. An unsubstituted carbamate could not be prepared. The mitosene thus synthesized differs from previously reported 1,2-disubstituted mitosenes, which are derived from the solvolysis of mitomycins, in that it has the opposite arrangement of oxygen and nitrogen substituents at the 1 and 2 positions. It showed antibacterial activities in disk-plate assays superior to those of cis-diacetylapomitomycin A and equivalent to those of certain 1-substituted mitosenes; however, it was less active than mitomycin A in these assays. It was inactive in inducing lambda-bacteriophage in Escherichia coli and inactive against P388 leukemia in mice.  In contrast, certain 1-substituted mitosenes were active in prophage induction and 2b and mitomycin A were active in both assays.


Synthesis and identification of the major metabolites of prazosin formed in dog and rat. The 6-O-demethyl and 7-O-demethyl analogues of the new antihypertensive drug prazosin [2-[4-(2-furoyl)-piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline hydrochloride] have been unequivocally synthesized via separate ten-step reaction sequences starting from isovanillin and vanillin, respectively. The 6-O-demethyl derivative was found to be identical with the major prazosin metabolite formed in dog and rat, while the 7-O-demethyl derivative was identical with another, less prevalent but significant metabolite. Two minor metabolites of prazosin, 2-(1-piperazinyl)-4-amino-6,7-dimethoxyquinazoline and 2,4-diamino-6,7-dimethoxyquinazoline, are also described. All four metabolites are less potent blood pressure lowering agents in dogs than prazosin but may contribute to its antihypertensive effect, since they account for a major portion of the administered dose.


Synthesis and biological activity of some new furan quaternary salts. A series of new N-(5-substituted 2-furfuryl)-N,N-dimethyl-N-aryloxyalkyl quaternary ammonium salts relating to general structure IV has been synthesized by reacting 5-substituted 2-(N,N-dimethylaminomethyl)furans IIa-d with appropriate aryloxyalkyl bromides III. The resulting compounds are tested for in vitro antimicrobial activity. A simpler synthesis of 5-nitro-2-(N,N-dimethylaminomethyl)furan (IId) involving the reduction of N,N-dimethyl-5-nitro-2-furamide (Ib) with diborane is described. A new compound, 5-bromo-2-(N,N-dimethylaminomethyl)furnan (IIc), is prepared in a similar way. Many of these compounds (22, 28, 34, 37-42, 44, and 45) indicate high activity against Staphylococcus aureus, Streptococcus faecalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa and are more active than nitrofurantoin, Compounds 22, 34 and 41 exhibit the highest in vitro antibacterial activity in the series. Some of these quaternary salts (22, 25, 37, 37-41, and 60) possess appreciable activity against Mycobacterium tuberculosis H37Rv. None of these compounds show significant antifungal activity. Eight compounds (18, 21, 22, 26-28, 32, and 34) have high in vitro antibacterial activity were inactive when tested for anthelmintic activity in rats against Nippostrongylus brasiliensis and Hymenolepis nana.


Syntheses and pharmacological activity of substituted imidazolidinethiones and thiomidazolines. A series of imidazolidinethiones and thioimidazolines was synthesized and tested for their effects on both forced and spontaneous motor activity as well as for their ability to raise the convulsion threshold. The proton NMR spectra for the thioimidazolines synthesized were unusual in that they showed a sharp singlet for the ring ethylene unit rather than the expected A2B2 pattern. The thioimidazolines, 5 and 7, were the most active CNS depressants and had the highest safety index. Significantly, the isomeric imidazolidinethiones, 8 and 9, were comparatively much less effective while being considerably more toxic.


A rapid, high-yield conversion of codeine to morphine. Brief treatment of codeine (1) in chloroform with boron tribromide has consiste-tly given morphine (2) in 90-91% yield after a simple isolation procedure. The yield and simplicity of operation in this method are vastly superior to those previously reported for this transformation.


An improved method for O-demethylation of codeine. The O-demethylation of codeine was effected by sodium propylmercaptide in dimethylformamide at 125 degrees C to afford morphine in 80% yield. Similar treatment of thebaine was unrewarding.


(2-Carboxy-1,4-dihydro-4-oxoquinolyl)oxamic acids and (2-carboxy-1,4-dihydro-4-oxobenzo[h]quinolyl)oxamic acids as antiallergy agents. A group of (2-carboxy-1,4-dihydro-4-oxoquinolyl)oxamic acids (5) containing the oxamic acid group in the 5,6, or 7 positions were synthesized and investigated for antiasthma activity as indicated by the passive cutaneous anaphylaxis (PCA) reaction in rats. Also synthesized and investigated were two (2-carbosy-1,4-dihydro-4-oxobenzo[h]-quinolyl)oxamic acids (9 and 10). Several of the compounds synthesized (viz. 5e, 5f, and 10) showed activity in the PCA test approximately 25 times that shown by disodium cromoglycate (1), as measured by the ID50 doses.


Synthesis and antiinflammatory activity of some 2-substituted 4- and 7-benzoxazoleacetic and alpha-methylacetic acids. 4- and 7-benzoxazoleacetic and alpha-methylacetic acids substituted in the 2 position with phenyl and substituted phenyl groups have been synthesized and tested on the carrageenan-induced rat paw edema assay. The only compound found to have significant activity, albeit of a low order, was 2-phenyl-alpha-methyl-7-benzoxazoleacetic acid.


Cannabinoids. 1. 1-Amino- and 1-mercapto-7,8,9,10-tetrahydro-6H-dibenzo [b,d]pyrans. A series of 1-amino- and 1-mercapto-7,8,9,10-tetrahydro-6H-dibenzo[b,d]pyrans was synthesized and subsequently evaluated in three rodent test systems for CNS activity. The structure-activity data generated indicate that, in general, a change of the 1-hydroxy group to an amine results in a retention of pharmacological activity but that a change to sulfur results in loss of pharmacological activity. Derivatization of the 1-amino group with various functions decreased the activity of the parent compound. For optimum potency, in all series, the 3-position alkyl side chain should be either 1,1- or 1,2-dimethylheptyl. With either the 1-hydroxy- or 1-amino-7,8,9,10-tetrahydro-3-(1,1-dimethylheptyl)-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (4c or 10c), preparation of the optically active antipodes did not lead to any great degree of spearation of activity. Both of the antipodes possess pharmacological activity as measured in these rodent test systems.


Butyrophenones from the isomeric 2-amino-5-phenylbicyclo[3.3.1]nonanes. The preparation of 5-phenylbicyclo [3.3.1]nonan-2-one is described starting from the ketal of 4-phenyl-4-(2-carbethoxyethyl)cyclohexan-1-one. The ketone was then taken on to the butyrophenone derivatives of endo-and exo-5phenylbicyclo[3.3.1]nonyl-2-amine. CNS screening results of these compounds are described.


Synthesis of cephalosporin-4-aldehydes.

Synthesis of cephalosporin-4-aldehydes. The first reported synthesis of cephalosporin-4-aldehyde derivatives has been achieved via Moffatt oxidation of the corresponding 4-hydroxymethylcephalosporins. The aldehyde 1 was converted into a number of polar derivatives, in particular the acrylic acid derivative 13 which is the 4-vinylogue of sodium cephalothin. None of the new cephalosporin derivatives possessed useful antibacterial activity.


Model studies of pyridoxal Schiff's bases. Coplanarity and intramolecular hydrogen bonding. The interactions between the pi cloud of the aromatic ring and the pi-electron pair of the imine double bond of aromatic oximes as model compounds of pyridoxal Schiff's bases have been studied by high-resolution carbon-13 magnetic resonance spectroscopy. The coplanarity and intramolecular hydrogen bonding have been determined by 13C-1H long range couplings. This detailed investigation of 13C-1H coupling also provides unambiguous proof of the existence of the "enol-imine" tautomers in chloroform and dimethyl sulfoxide solutions. The tautomerism between the "enol-imine" and "keto-enamine" is discussed.


Synthesis and biological activity of beta-melanotropins and analogues. Synthetic [Arg8]-, [Gly10]-, and [Phe12]-beta cl-MSH and beta p-MSH have been prepared by the solid-phase method. Their lipolytic and melanotropic activities have been compared to each other and with those of previously synthesized beta cl-MSH, [formyl-Trp12]-beta cl-MSH, and beta b-MSH. Replacement of Bln8 of beta cl-MSH by Arg causes a greater decrease in melanotropic activity than lipolytic activity. However, when Phe10 of beta cl-MSH is replaced by Gly, both activities are destroyed. Alteration or replacement of Trp12 of beta cl-MSH by formyl-Trp or Phe has little effect on the melanotropic activity while the lipolytic activity is greatly decreased. Replacement of Ser2 of beta b-MSH by Glu induces a three fold increase in melantropic activity but it does not affect the lipolytic activity.


Structure-activity relationship in cinnamamides. 2. Synthesis and pharmacological evaluation of some (E)- and (Z)-N-alkyl-alpha, beta-dimethylcinnamamides substituted on the phenyl group. Several (E)- and (Z)-N-alkyl-alpha,beta-dimethylcinnamamides variously substituted on the phenyl group were synthesized from their corresponding acids and characterized through their NMR spectra. The compounds were tested to determine the relationship existing between their action on the CNS and the activity exhibited by the corresponding amides unsubstituted on the phenyl, previously studied. Substitution with the same group always had the same effects on the biological activity of the (E)-N-alkyl-alpha,beta-dimethylcinnamamides selected; these effects mainly regarded anticonvulsant activity which is the most noteworthy action of these compounds. This activity was reduced by electron-donating substituents and increased by electron-withdrawing ones. In the Z series the p-phenyl substitution with a halogen reduced or suppressed the CNS stimulant activity exhibited by the parent compounds.


Studies on vitamin D (calciferol) and its analogues. 10. Side-chain analogues of 25-hydroxyvitamin D3. A homologous series of side-chain analogues of 25-hydroxyvitamin D3 (25-hydroxycholecalciferol) in which the length of the side chain is modified while maintaining its characteristic tertiary hydroxyl moiety has been synthesized. The following five analogues have been prepared and characterized: pentanor-25-OH-D3 (2a), trinor-25-OH-D3 (2b), dinor-25-OH-D3 (2c), nor-25-OH-D3 (2d), and homo-25-OH-D3 T2e). Biological assays in vivo of intestinal calcium absorption and bone calcium mobilization in the chick of the five analogues revealed that the homo analogue 2e exhibited a significant biological response relative to the -D (vitamin D3) control. Compared to the natural vitamin D3, 2e is as active in its ability to mobilize bone calcium and is about half as effective in stimulating intestinal calcium transport. The remaining analogues (2a-d) exhibited no significant activity in either assay, although the nor analogue 2d was previously observed to exhibit antimetabolite activity.


Effect of sulfur substitution for the noncarbonyl oxygen in hemicholinium-3 and acetyl-seco-hemicholinium-3. Synthesis, biological activity, and structure-toxicity relationships. As a continuation of our efforts to develop and study inhibitors which act presynaptically on neuromuscular function, sulfur analogues of hemicholinium-3 (HC-3, 1) and acetyl-seco-hemicholinium-3 (AcHC-3, 3) were prepared. In each case sulfur is substituted for the noncarbonyl oxygen in HC-3 (1) and AcHC-3 (3). As expected on the basis of conformational differences between acetylcholine and acetylthiocholine both of the thio analogues are produced in the seco form and do not cyclize spontaneously or when subjected to aqueous, acidic conditions up to 100 degrees C. Both compounds are stable in aqueous pH 7.4 solutions at 37 degrees C and in slightly acidic D2O solutions for more than 24 h. While thio-seco-hemicholinium 3 (11) is stable in the presence of acetylcholinesterase and butyrylcholinesterase in H2O at pH 7.4, acetylthio-seco-hemicholinium-3 (12) reacts within seconds to form the hemiacetal form of thiohemicholinium-3 (16). Mouse toxicity studies (LD50) indicate that while 12 is approximately as toxic as HC-3 (1) and AcHC-3 (3), 11 is 226 times less toxic. As in the studies with 1 and 3, mice were protected from 11 by choline and slightly by neostigmine. It is of interest, however, that almost equal and intermediate protection against 12 was afforded by choline and neostigmine. Structure-toxicity relationships of 1,3,11, 12, and 16 are discussed.


Dibenz[b,e]oxepinalkanoic acids as nonsteroidal antiinflammatory agents. 1. 6,11-Dihydro-11-oxodibenz]b,e]oxepin-2-acetic acids. A series of 6,11-dihydrodibenz[b,e]oxepin-2-acetic acids has been evaluated for both antiinflammatory and analgetic activity in the carrageenan paw edema and phenylquinone writhing assays. The requirements for optimal activity in this series appear rather specific: (a) an unsubstituted 6,11-dihydrodibenz[b,e]oxepin nucleus and (b) a carbonyl group in the 11 position. One derivative, 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid (11), has been selected for further study.


Synthesis and kinetic evaluation of S- and N-substituted cysteinylglycines as inhibitors of glyoxalase I. Eight S- and N-substituted L-cysteinylglycines were prepared by condensation of S-benzyl-L-cysteinylglycine or S-(p-bromobenzyl)-L-cysteinylglycine with glutaric anhydride, succinic anhydride, or the appropriately blocked and activated amino acids. In contrast to the previously prepared S-substituted glutathiones, all of the title compounds exhibited noncompetitive inhibition of yeast glyoxalase I. A kinetic evaluation under Yonetani-Thorell conditions established the existence of two binding sites on the glyoxalase I enzyme.


Synthesis and antiinflammatory and hypnotic activity of 5-alkoxy-3-(N-substituted carbamoyl)-1-phenylpyrazoles. 5-Alkoxy-3-(N-substituted carbamoly)-1-phenylpyrazoles were prepared and tested for antiinflammatory and hypnotic activity. Four compounds showed antiinflammatory activity and three possessed hypnotic properties.


Ethics/human values education in the family practice residency. Courses in medical ethics (and other humanities) have been introduced into the curricula of many medical schools, but formal attention to ethics and the value dimensions of medical practice have been largely absent from a crucial phase of medical education and socialization--the residency. In this paper the scope of ethics is identified in terms of the normal professional experiences of a family physician. The author describes in detail the six educational objectives and the instructional methods that form the basis of the ethics/human values curriculum in the Department of Family Practice residency program, Medical University of South Carolina.


Observations on a rural health manpower project. The Rural Externship Program was developed by the Lakes Area Regional Medical Program in conjunction with the State University of New York at Buffalo and the health professionals of western New York and northwestern Pennsylvania. It was designed to encourage health science students to practice in a rural area following graduation. This interdisciplinary program provides health science students with an eight-week summer living-working experience in a rural environment, supervised by practitioner-preceptors. The intent is to develop their appreciation of rural health care and life-styles. Since the summer of 1970, 240 students have participated. This paper describes and discusses the project and presents measures of the externs' changes in attitudes toward rural practice which occurred after they participated in the program. A survey of externs who have graduated shows the effect of the program on their decision to locate. Of the 61 externs contacted, 55 percent indicated that they were in rural practice, and 53 percent of the latter indicated that their experience in the Rural Externship Program was an important factor in their decision to practice in a rural area.


An independent study program within a medical curriculum. The Independent Study Program (ISP) at the University of Rochester School of Medicine and Dentistry is described and its objectives are specified. Experimental (ISP) students and two matched (non-ISP students from the same class) groups were studied in two successive second-year medical school classes. Data were gathered only post-ISP for the first class and both pre- and post-ISP for the second. The data included measures of achievement, personality characteristics, and academic-professional experience. Experimental and control groups were not significantly different on achievement and academic-professional measures but did differ on certain measures of personality and values. The groups also differed on their evaluations of curriculum and reports of study habits. Fache ISP program was considered, and it was found that the ISP is less expensive of faculty time than is the regular curriculum but involves higher nonfaculty costs.


The medical record review and resident education. The purpose of this paper is to consider the medical record review in resident education. With data obtained from mother-infant pairs during the puerperium as an example, three possibilities for using this method are discussed: (a) as a means by which faculty and trainees, working together, could specify objectives relating to patient care and trainee competence; (b) as a tool in assessing both the clinical progress of trainees over time and the effectiveness of a teaching program; and (c) as a way of helping both faculty and trainees become more than expert "doers," that is knowledgeable physicians critically assessing the effects of medical science applied to the care of their patients.


The 'new' medical student: another view. In this paper the authors examine some evidence concerning admission of a "new" type of student to medical school. Such students have been described as more socially concerned and committed to reform of the health care system than students of the 1950s. The hypothesis was that the new type of student was a temporary phenomenon, influenced by transitory sociopolitical events which had little impact on the institutionalized processes of medical education. Data consisted of responses to selected attitude statements by successive entering classes of medical students in one school of medicine from 1970 to 1975. Data were collected a second time from graduating seniors in 1974 and 1975 (entering classes of 1970 and 1971). The same information was collected from physician preceptors in 1975. Results suggest partial support for the hypothesis of decreasing liberalism among entering freshman after 1973, increasing conservatism among graduating seniors, and a high level of conservatism among physicians. Some exceptions to these trends are noted.


Family medicine and primary care: trends and student characteristics. Using data from a longitudinal study of medical students at Jefferson Medical College, the authors analyzed trends in senior student interest in primary care specialties between 1971 and 1975 and selected background characteristics and performance levels of students choosing family medicine compared with those in other specialties. The proportion of students interested in family medicine residencies increased from 6 to 17 percent in the study period. The academic performance of students selecting family medicine was equal to or better than that of those in all other specialties except internal medicine. Smaller proportions were interested in teaching and research than those in other specialties, and larger proportions intended to work in communities of 100,000 population or less.


Specificity of antitumor immune reactions mediated by xenogeneic immune RNA. Evidence that xenogeneic immune RNA (I-RNA) mediated specific cytotoxic immune responses against human tumor-associated antigens was obtained from in vitro studies in two autologous melanoma systems. In these systems, malignant melanoma target cells, matching normal fibroblast target cells, lymphocyte effector cells, and melanoma and normal skin tissue used to immunize RNA donor animals were derived from the same autochthonous hosts. When incubated with autologous lymphocytes, I-RNA extracted from the lymphoid organs of donor animals immunized with melanoma tissue mediated immune reactions against autologous melanoma target cells in vitro. I-RNA from animals immunized with normal skin tissue from autochthonous hosts did not increase the cytotoxicity of autologous lymphocytes for autologous melanoma cells. Using autologous fibroblasts as target cells, we detected no increase in cytotoxicity when autologous lymphocytes were incubated with RNA from animals immunized either with melanoma tissue or normal skin tissue from the autochthonous host. By contrast, when allogeneic lymphocytes were used as effector cells, RNA extracted from animals immunized either with melanoma tissue or normal skin mediated cytotoxic immune reactions against melanoma target cells and normal fibroblast target cells derived from the same patient.


Modification of hydroxyindole-O-methyltransferase activity in experimental pineocytomas induced in hamsters by a human papovavirus (JC). Hydroxyindole-O-methyltransferase (HIOMT) (HIOMT) activity was studied and compared in 7 pineal tumors (pineocytomas) induced in vivo in Syrian hamsters after postnatal inoculation with strains of human papovavirus (JC). Levels of tumor HIOMT activity were correlated with degree of cytologic differentiation of the pineocytes of each neoplasm, as judged from electrom micrographs. The relative capacities of the HIOMT in the tumors to methylate three related substrates gave similar ratios in the individual tumors and were not different from those of HIOMT in normal hamster pineal glands.


Epidemiologic pathology of gastric ulcer and gastric carcinoma among Japanese in Hawaii. This case-control study of Hawaiian Japanese indicated that gastric ulcer in the proximal portion of the pyloric antrum has features similar to those of gastric cancer. Such ulcers occurred at sites most frequently and most severely affected by intestinal metaplasia, although metaplasia tended to be more extensive with cancer than with ulcer. Metaplastic mucosa was more vulnerable to the action of pepsin and acid than was normal mucosa. The risk of ulceration would rise when a sufficiently lagrge area of the antrum was intestinalized and when the corpus continued to produce significant quantities of these substances. This study showed a strong association between salt intake, ulcer, and metaplasia. Significant but less dramatic associations were demonstrated between metaplasia and the use of traditional Japanese foods and smoking. The question was raised as to whether salt promotes ulceration or whether it potentiates the action of a mutagen that causes intestinal metaplasia.


Brief communication: Possible increased risk of lung cancer among beauticians. Death certificates of residents of Alameda County, California (1958-1962) with any mention of cancer were examined to determine the risk of cancer among beauticians. Twenty-four female beauticians were found among 3,460 adult females who died with cancer; four female beauticians were found in a sample of 1,000 females who died from causes other than cancer. The case and control certificates were matched for age and race. The resulting odds ratio (i.e., approximate relative risk of a cancer death) was slightly elevated (1.73) but was not statistically significant. Although a possible increase in the risk of cancer was found for several organ sites, the increase in risk for lung cancer was most striking. Analysis of pairs of death certificates, in which female controls dying of causes other than cancer were matched to female lung cancer cases for age, race, and date of death, yielded an odds ratio of 6.0. This finding suggested that the risk of lung cancer may be substantially increased among female beauticians.


Putrescine-promoted mutual adhesiveness of HeLa cells from density-inhibited suspension cultures. The mutual adhesiveness of HeLa cells harvested from density-inhibited suspension cultures was significantly enhanced by the addition of putrescine. No significant effect was observed on the adhesiveness of HeLa cells harvested from fast-growing suspension cultures. The effect on density-inhibited cells was abolished by previous treatment of the cells with neuraminidase.


High resolution proton magnetic resonance spectral characteristics of water, lipid, and protein signals from three mouse cell populations. The high resolution proton magnetic resonance spectral properties of three C57BL/6J mouse cell populations (EL4 ascites tumor cells, normal spleen leukocytes, and normal erythrocytes) were investigated. Packed cell pellets were investigated at 100 MHz and 13.56 MHz. The 100-MHz spectra contained identifiable non-water proton resonances as well as the water resonance. The major non-water resonances from the EL 4 cells and normal leukocytes resembled resonances from lipid extracts, whereas the non-water resonances from erythrocytes resembled resonances from hemoglobin solutions. The reciprocals of the water proton spin-lattice relaxation times were roughly proportional to the total sample water content at both 100 and 13.56 MHz. The estimated slopes of these plots were frequency-dependent. The water proton relaxation rate generally increased with increasing total protein content of the packed cell pellets.


Concanavalin A receptors on the surfaces of human breast cancer cells in organ culture. The binding of concanavalin A (Con A) at the free apical membranes of surface tumor cells in human breast cancer explants grown in organ culture was studied cytochemically with horseradish peroxidase (HRP) as a marker. On the cell membranes in aldehyde-fixed explants or explants exposed to Con A at 4 degrees C, a continuous label covered the entire free surface of the cell, which indicated the dispersed distribution of Con A binding sites. The binding of Con A at 20 degrees C resulted in discontinuous label of the cell surface, with gaps of unlabeleled membrane and partial endocytosis of the label. Incubation at 37 degrees C, following the binding of Con A and HRP at both temperatures, induced more extensive, incubation time-dependent discontinuities of the surface label that led to complete disappearance of the label from the surface and its eventual endocytosis. Con A was topically stablized on the surface only in those regions where two membranes or a different part of a folded membrane were in close contact. No differences were found in the binding, redistribution, and internalization of bound Con A among the various tumors studied.


Ultrastructure of spontaneously differentiated human malignant melanocytes cultured from primary tumors. Ultrastructural study of early subcultures and established lines of human malignant melanocytes derived from primary tumors showed that melanocytes passed through a phase of dedifferentiation during which they took on a fibroblast-like appearance; then they had a phase of spontaneous redifferentiation. The fibroblast-like cells were characterized by the presence of network-like organelles in their cytoplasm, and the melanocytes by melanosomes.


Identification of a tumor-associated antigen in cervical carcinoma by two dimensional (crossed) immunoelectrophoresis. Two-dimensional immunoelectrophoresis was used to evaluate 8 cervical cancer speciments, 11 other gynecologic tumors, and 5 specimens of normal cervix. Antigens were water-soluble tissue extracts and antisera prepared in rabbits. When tested against antisera to cervical cancer, cancer antigens showed 14-17 precipitin lines whereas normal cervix showed 10-16. A single heavy heterogeneous precipitin line with an electrophoretic mobility of 0.58 relative to bovine albumin was observed in all cervical cancer specimens but not in normal cervical or other tumor specimens. Further evidence for the uniqueness of this antigen was sought by enhancement (addition of another antigen to the first phase of electrophoresis which increased the size of common peaks) and suppression (addition of another antiserum to the second phase, whereby the peak size of components to which both sera have antibody was decreased). The specific precipitin line was neither suppressed by the addition of antisera to normal tissue nor enhanced when normal tissue antigen was added to the tumor antigen preparation. More conclusively, adsorption of the tumor antiserum with normal tissue had no effect on the unique tumor-associated precipitin line, whereas all other precipitin lines were removed. This antigen was common to other cervical tumors because enhancement was demonstrated with three other cervical tumor specimens. The identification of a distinct and separate antigen associated with cervical carcinoma will permit further characterization and possible development of immunodiagnostic methods.


Chromosomes and causation of human cancer and leukemia. XX. Banding patterns of primary tumors. Banding techniques were used in the study of the chromosomes of primary tumors from 16 patients with various types of cancer. The initial analysis with conventional Giemsa staining revealed chromosome abnormalities in 13 of the 16 tumors. Eleven of these 13 tumors and 2 of the 3 with normal karyotypes were reexamined with Q-, G-, and C-banding techniques: The 2 tumors were conventionally stained normal karyotypes were found to have no abnormalities. Nine of the tumors wre characterized by numerical changes only and 4 by both numerical and structural abnormalities. In 11 tumors, excessive chromosomes, identified with banding techniques, were usually found in the following groups (number of tumors involved is shown in parentheses): No. 5 (5), No. 8 (6), No. 11 (5), no. 13 (5), and No. 21 (5). The primary tumors examined had hyperdiploid modes; only 4 of these tumors contained marker chromosomes, as opposed to the high frequency of markers in metastatic cancer cells and the presence, usually, of high polidy (near-triploidy or near-tetraploidy). The data suggested that the karyotypic changes in primary cancers consist primarily of numerical changes (hyperploidy), rather infrequent appearance of marker chromosomes, and, when present, only 1 or 2 markers.


Histologic features and observational variation in cerebellar gliomas in children. Variation existed in the recognition of histologic features commonly used in the evaluation of cerebellar gliomas of childhood. Some histologic features (e.g., perivascular pseudorosettes, leptomeningeal deposits, and calcification) were more reliably observed than were others (e.g., Rosenthal fibers, cell density, and hypervascularity). Knowledge of which features tend to have greater observational variation may lead to improved definitions, less reliance of these features in clinical decisions, further studies of the potential sources of the variation, and guidelines for minimizing observational variation.


Advantages of cryoperserved lymphocytes for sequential evaluation of human immune competence. I. Mitogen stimulation. Human lymphocytes were harvested from normal volunteer donors and cryopreserved. Various concentrations of dimethyl sulfoxide and human serum in the cryoprotective media were evaluated to optimize the recovery and function of viable cells. Multiple samples were then drawn from volunteers over a number of days, processed individually, and used in mitogen stimulation assays. These cells were also cryopreserved, immediately thawed, and cultured simultaneously with the fresh cells. In all instances fresh and cryopreserved lymphocytes exhibited similar levels of stimulation by mitogens. Cryopreserved cells from these sequential bleedings were then recovered and cultured simultaneously in mitogen stimulation assays. The results obtained in these assays with cryopreserved cell had less day-to-day variagion than those with cells cultured individually. Coefficients of variance of individual cultures of mitogen stimulation assays were reduced from 26-59% to 5-17% by use of cryopreserved cells. The findings suggested that use of cryopreservation techniques decreases the variability of cellular immune assays and thus alows more accurate longitudinal study of the immune competence of patients.


IgG4 subclass in malignant melanoma.

IgG4 subclass in malignant melanoma. Three hundred and ninety-seven sera from 185 melanoma patients were studied. These sera were classified into three groups according to stage of disease. An alteration in the level of the IgG4 subclass was found. It was related to the dissemination of disease. The percentage of abnormalities (either increased or decreased levels of IgG4) was more frequent in patients with stage II and III diseases (55 and 53%, respectively) than in patients with stage I(19%). The higher frequencies of high titers of IgG4 were essentially detected in advanced disease. The biologic significance of the increase of IgG4 in melanoma remains obscure. The increase may be related to the development of facilitating antibodies of the IgG4 subclass.


Differences in breast cancer between Japan and the United States. Two surgically treated series of 375 Japanese and 352 American women with breast cancer were compared with respect to overall survival and recurrence-free survival rates. Recurrence-free survival rates in Japanese women were superior to those in American women. Overall survival rates were also better in Japanese women, although the difference was not nearly as great as for recurrence-free survival. This seemed due to an improved survival after metastasis in the American women. In analyzing recurrence-free survival rates by age, we found that Japanese women 40 to 54 years old had better recurrence-free survival rates than other age subgroups. This age advantage was not observed in American women. For other age groups, there was relatively little advantage in survivorship in the Japanese series.


Absence of HeLa cell contamination in 169 cell lines derived from human tumors. Numerous cell lines derived from human tumors are not HeLa contaminants. Of 192 lines established in this or other laboratories, 169 lines were found to be G6PD type B. Twenty-three lines were type A as HeLa; three of these were of Negroid origin. There is reasonable doubt that the remaining 20 lines will all be shown to be confounded with HeLa.


Enzymatic susceptibility and spontaneous release of human melanoma tumor-associated antigens. A chimpanzee anti-human melanoma antiserum was used to study the enzymatic susceptibility and spontaneous release into tissue culture medium of human melanoma tumor-associated antigens (TAA). Limited proteolytic digestion of melanoma cells with trypsin or with pronase rendered these cells refractory to lysis by the chimpanzee antiserum and complement. Longer periods of incubation of higher concentrations of enzyme caused an increased sensitivity to lysis. Digestion of melanoma cells with neuraminidase apparently exposed antigens reactive with natural antibodies in rabbit complement because cells so treated had a marked increase in sensitivity to cytolysis. Absorption of the complement with either neuraminidase-treated human melanoma cells or washed human spleen cells prior to its use in the cytotoxicity assay removed this activity. When absorbed complement was used, neuraminidase had no noticeable effect on the expression of malanoma TAA. These results suggest that proteolytic digestion of melanoma cells may prove to be a useful means of solubilizing TAA. The spontaneous release of melanoma cell membrane TAA was studied. Protein precipitated by (NH4)2SO4 from four of six samples of tissue culture medium used to feed malanoma cell lines contained significant antigenic activity compared to a control "antigen" preparation, whereas one preparation contained only limited TAA activity. One melanoma cell line that apparently failed to release TAA into the culture medium had previously become nonreactive with the chimpanzee antiserum. From these data, we conclude that melanoma cells growing in tissue culture rapidly release large amounts of TAA into the culture media and, as a result, the spent culture medium may be a good source for obtaining TAA for further study. The significance of these results is discussed.


Chromatographic and electrophoretic analysis of an antigen in Hodgkin's disease tissue cultures. An antigen in tissue cultures derived from Hodgkin's disease tumors was investigated by polyacrylamide gel electrophoresis, column chromatography, and isotopic antibody techniques. Fourteen long-term, serially passaged monolayer cultures prepared from tumor nodules of Hodgkin's disease in the spleen were studied; 11 monolayers derived from normal adult spleen and human fetal spleen and thymus were used as controls. Cell-free medium from Hodgkin's disease and normal cultures were centrifuged, and the pellet fractions were sedimented in a discontinuous sucrose gradient, solubilized with dodium dodecyl sulfate, and labeled with radioiodine. Gel filtration and electrophoresis revealed a component in samples prepared from medium of Hodgkin's disease cultures that was not observed in medium from normal cultures. An antiserum made in rabbits against this component reacted by radioiodine-labeled antibody assay with an antigen on the surface on cells from Hodgkin's disease cultures that was present in very small amounts, or in a cryptic state, on normal cultured cells. This antigen, intimately associated with propagation of cells obtained from the tumor in vitro, was not demonstrable in noncultured Hodgkin's disease tissue...


Alteration of cell-surface antigenicity of the mouse plasmacytoma. II. Lack of correlation between synthesis of myeloma protein and alteration of surface antigen. The correlation between the changes of immunoglobulin synthesis and the surface antigenicity was analyzed by cytotoxicity and quantitative antibody-absorption tests with the cells of immunoglobulin-producing and nonproducing mouse plasmacytoma. IgA-synthesizing BLAB/c plasmacytoma 58-8 and the non-IgA-synthesizing variant of the 58-8 (nonproducer) were killed with rabbit anti-58-8 plasmacytoma cell antiserum, C3H/He anti-BALB/c spleen cell antiserum, and (C57BL/6 X DBA/2)F1 anti-BALB/c plasmacytoma cell MOPC-31C antiserum plus complement, only when the cells were pretreated with pronase. Quantitative absorption tests revealed that the nonproducer, 58-8 had the same amount of plasmacytoma antigen of 58-8 and PC.1 antigen, and a greater amount of H-2d antigen, as did producer 58-8. The same analysis was carried out for the C3H mouse plasmacytoma X5563, which has an M-component of IgG2a. The nonproducer X5563 had a greater amount of H-2k antigen and a smaller amount of the plasmacytoma antigen of X5563 than did the producer X5563. No detectable PC.1 antigen was observed at surfaces of the producer and the nonproducer X5563 cells.


Galactosyltransferase activity in metastasizing and nonmetastasizing rat mammary carcinomas and its possible relationship with tumor cell surface antigen shedding. In order to study the mechanism of tumor cell surface antigen shedding, galactosyltransferase levels were compared in 5 spontaneously metastasizing and 3 nonmetastasizing rat mammary tumors. The enzyme activity both with or without exogenous acceptors was higher in the metastasizing group. This difference did not seem to be due to the variation in levels of degrading enzymes such as pyrophosphatase or beta-galactosidase found in these tumors. Little difference in the biochemical properties of the enzyme was found between the two groups. Most of the enzyme activity (60-70%) was recivered in the microsomal frctosyltransferase was assayed in "purified" plasma membrane fractions, 70% of the activity was associated with the plasma membrane vesicles, in which the enzyme was enriched by factors of 10-40. The number of galactose acceptor sites on the plasma membranes increased in parallel to the metastasizing capacity, indicating the presence of larger numbers of incomplete glycopeptides on their cell surfaces. These findings seemed to indicate that the greater turnover of glycoprotein in the spontaneously metastasizing tumor cell surface was caused by the shedding of surface antigens into the systemic circulation of the host.


Absorption and protein binding of N-2-fluorenylacetamide and its metabolites in the bladder of rabbit. To assess the reactivity of a bladder carcinogen, the absorption by the rabbit (male New Zealand White) bladder mucosa of N-2-acetylaminofluorene (AAF), N-hydroxy-2-acetyl-aminofluorene (N-OH-AAF), and the N-O-glucuronide of AAF (N-OGI-AAF), as well as binding to the protein and RNA of bladder mucosa, was measured in vivo and in vitro. Mucosal pieces incubated for 3 hours in medium containing a carcinogen demonstrated that the fluorene nucleus of both AAF and N-CH-AAF bound equally with cellular proteins, while N-OGI-AAF binding was lower. In the presence of an excess of beta-glucuronidase, however, N-OGI-AAF showed binding equivalent to its metabolic precursor. After a 3-hour instillation into the bladder lumen of radioactive carcinogens suspended in urine in vivo, transmural absorption of AAF and N-OH-AAF (90%) was substantial, while N-OGI-AAF was absorbed less (55%). The renal excretion during this period varied from 18 to 52% of the instilled radioactivity. There was little reactivity of these carcinogens with the mucosal RNA, both in vivo and in vitro. The metabolism of N-OH-AAF and N-OGI-AAF was such, both in vitro and in vivo, that the acetyl group was not included in the final protein-carcinogen complex in what appeared to be an enzyme reaction.


Organ distribution of Corynebacterium parvum labeled with iodine-125. Administration of iv, ip, single sc, multiple sc, and footpad injections of [125I]Corynebacterium parvum in mice revealed different patterns of radioactive vaccine distribution in various organs. High deposition and retention were found in the liver, spleen, and gastrointestinal tract and less in the lungs, kidneys, thymus, and bone marrow. Control animals given 125I showed very rapid clearance of the isotope and no retention in the organs. The pattern of distribution of [125I]C. parvum could be useful when protocols for clinical trials are designed.


Enhancement of urethan tumorigenesis in mouse lung by butylated hydroxytoluene. Intraperitoneal injection of the antioxidant butylated hydroxytoluene (BHT) produces cell proliferation in mouse lungs within 2-4 days. We examined whether the presence of an increased number of proliferating lung cells would influence urethan tumorigenesis. Male Swiss-Webster mice were treated with 1 mg urethan/g before, during, or after BHT-stimulated cell growth in the lung. The number of pulmonary tumors found 13-15 weeks later was not different in BHT-treated mice compared to that in controls. On the other hand, repeated stimulation of cell growth after urethan treatment enhanced tumorigenesis. Male Swiss-Webster and A/J mice were given a single dose of urethan (1 mg/g) and, beginning 7 days later, weekly injections of BHT or corn oil. Repeated injections of BHT significantly increased the yield of lung tumors in both strains. Weekly injections of BHT into mice pretreated with 0.9% NaCl reduced the number of spontaneous pulmonary adenomas.


Histopathology of preneoplastic and neoplastic lesions of the esophagus in BUF rats ingesting diethylnitrosamine. Carcinomas developed in the mucosa of the esophagus in BUF strain rats ingesting diethylnitrosamine in the diet. The stages of hyperplasia, hyperplastic nodules, small carcinomas, and large, well-developed carcinomas were observed. Carcinomas, which were well differentiated, poorly differentiated, or undifferentiated, invaded the adjacent tissue but did not metastasize.


Comparison of three isotopic assays of cell-mediated cytotoxicity against mouse tumor cells. I. Basic parameters: baseline controls, target cells, and methods of calculation. We compared three ddifferent isotopic assays of cell-mediated cytotoxicity under identical test conditions: 51Cr, 125iododeoxyuridine, and [3H]proline release assays. We made these comparisons both in a syngeneic mouse tumor system and an allogeneic system. It was found that several parameters could affect considerably the results obtained with these tests, such as: baseline controls, preparation of target cells, and methods of calculation. In comparison of three different baseline controls, the normal control gave the most consistent results, whereas the other two controls (autologous and medium controls) gave varying results depending upon the condition of the target cells. For use as target cells, established tissue culture cells were usually superior because of a much lower spontaneous release of the isotope, when compared to fresh ascites tumor cells or short-term tissue culture cells. The advantage of established culture cells was particularly noted in isotopic assays with prolonged incubation (20-40 hr). In addition, the methods used for calculation were also shown to affect the apparent outcome of the results.


Comparison of three isotopic assays of cell-mediated cytotoxicity against mouse tumor cells. II. Sensitivity and specificity of the assays and characteristics of effector and sensitizing cells. Three assays of cell-mediated cytotoxicity in mice, involving release of either 51Cr (CRA), 125iododeoxyuridine (IRA), or [3H]proline (PRA), were compared under identical test conditions. Experiments were performed with effector cells from mice immunized with FBL-3 tumor cells, a syngeneic Friend virus-induced leukemia, or with allogeneic normal spleen cells. With established tissue culture cells as targets, similar results were obtained in all three assays. The cytotoxicity produced by cells from in vivo-immunized mice and the induction of cytotoxicity in vitro were T-cell-dependent. When short-term culture target cells were used, the IRA gave a more selective pattern of cytotoxicity than did the other two assays. However, when remaining target cells at the end of the assay were treated with trypsin, higher levels of 125iododeoxyuridine (125IUDR) release were seen, and the results were then comparable to those in the CRA and PRA. These results indicated that 125IUDR, a nuclear label, could only be released after lysis of cells. In contrast, 51Cr or [3H]proline, which are cytoplasmic labels, could also be released from damaged but unlysed cells. These fundamental differences could give different results in these assays, which could determine their correlation with in vivo transplantation immunity.


Biologic analysis of fetal MRC rat lung epithelial cells treated with 3-methylcholanthrene in culture: premalignant and malignant stages. Fetal MRC rat lung primary cell sheets, in which reconstruction of bronchial tissue occurred, were treated with 3-methylcholanthrene (MCA) to examine the carcinogen's biologic effect on lung epithelial elements. Cloned lines were established by subculturing of epithelial outgrowths from primary cell sheets induced by MCA treatment. Newborn MRC rats received an sc injection of these cells to assess their tumorigenicity. These MCA-treated cells at the initial stages of culture temporarily formed benign epithelial structures in scar tissue that developed from an sc injection and then regressed. The epithelial structures were roughly classified as tubular and squamous epithelium. The differentiation of cells in vitro was observed as either a type of lamellar keratinization of the premalignant squamous cell form or a type of cyst formation by premalignant secreting epithelium. With further cultivation, six of 24 cell lines became malignant and, when injected into the animal, induced carcinomas. Qualitative changes in the in vitro differentiation of cells were observed in cell lines that formed squamous cell carcinomas. These changes included spherical keratinization (pearl formation) and single-cell keratinization. MCA treatment induced various premalignant alterations in lung epithelial cells, and the subsequent malignancy seemed to stem from these initial premalignant changes.


Carcinogenic effects of diisopropanolinitrosamine in Sprague-Dawley rats. The carcinogenic effects of diisopropanolinitrosamine (DIPN) were tested in Sprague-Dawley rats and were then compared with results produced earlier by the same substance in Syrian hamsters. In addition to the similarities, several differences were noted; for example, DIPN caused pancreatic tumors in all the hamsters, but only 1 pancreatic tumor was observed among the 150 rats. Administration of DIPN to rats led to the development of neoplasms in the nasal and paranasal cavities, lungs, thyroid gland, esophagus, liver, and kidneys. The highest tumor incidence in rats was in the nasal cavities. Almost all the pulmonary neoplasms were malignant and were usually squamous cell carcinomas. We found a 15.4-50% incidence of malignant tumors of the thyroid gland; we also noted that thyroid neoplasms occurred at almost the same rate (36.4-50%) in the groups given 1/5, 1/10, and 1/20 the median lethal dose.


Tumor-associated antigens in H-2 hemizygous isoantigenic variants of a somatic cell hybrid, derived from the fusion of a 3-methylcholanthrene-induced sarcoma and a mammary carcinoma. To check the suggestion that 3-methylcholanthrene-induced, sarcoma-associated, tumor-specific transplantation antigens (TSTA) could be modified H-2 antigens, reciprocal isoantigenic variants derived from TA3Ha/MSWBS hybrid cells were examined. This hybrid was produced by the fusion of the TA3Ha ascites carcinoma (H-2a) and the 3-methylcholanthrene-induced MSWBS ascites sarcoma (H-2s). MSWBS expresses a strong TSTA capable of inducing a rejection reaction in the syngeneic A.SW host. The genetic determinants of the H-2 complex are known to be localized on chromosome No. 17. TA3Ha contributes two normal, telocentric chromosomes No. 17 to the hybrid. In contrast, both chromosomes No. 17 of MSWBS are localized on readily identifiable translocations (17/1 and 17/M1). We have previously shown that the chromosomes No. 17 of one parental strain or the other (but not both) can be eliminated from the hybrid upon selective passage in the opposite parental strain. The present studies showed that the two strain A-compatible variants that have lost the sarcoma-derived 17-chromosomes still contained the same TSTA as the two reciprocal strain A.SW-compatible variants that have lost the mammary carcinoma-derived H-2 chromosomes. These findings argue against the possibility that methylcholanthrene-induced TSTA is a modified form of H-2 or that its structural determinants(s) is localized on chromosome 17.


Kinetics of nitrosamine formation in the presence of micelle-forming surfactants. The nitrosation of dihexylamine by nitrite was catalyzed in the presence of micelles of the cationic surfactant decyltrimethylammonium bromide. Rate enhancements up to 800-fold were observed. The magnitude of the rate enhancements was dependent on the amine structure, particularly the alkyl chain length. Rate enhancements for nitrosation were also observed in the presence of micelles of phosphatidylcholine and Triton X-100. We present a mechanistic interpretation of these catalytic effects.


Effects of butylated hydroxyanisole on the metabolism of benzo[a]pyrene by mouse liver microsomes. The metabolites of tritium-labeled benzo[a[pyrene (BP) extracted from liver microsomes prepared by butylated hydroxyanisole (BHA)-fed and control female A/HeJ mice were analyzed by high-pressure liquid chromatography. There was an overall decrease in diol formation as well as an increase in the phenol formation in microsomal incubations from the BHA-fed mice compared to the controls. The dione region, when analyzed together with the BP-4,5-oxide peak, showed an overall decrease with BHA feeding. The BP-4,5-oxide was isolated and identified. It was present in both the BHA-fed and control microsomal incubations but was substantially reduced in the former. BP-9,10-oxide and BP-7,8-oxide were not directly demonstrated. Data based on summation of diols and phenols resulting from the breakdown of these oxides indicated that they were present in reduced amounts in the microsomal incubation from BHA-fed mice. The 3-hydroxybenzo[a]pyrene (3-HOBP) was the major metabolite in both microsomal incubations and was present in a significantly higher percentage in the incubations from the BHA-fed mice when compared to control mice. Thus BHA resulted in two metabolic alterations which could result in its exerting an inhibitory effect on BP-induced carcinogenesis. The first was an increase in 3-HOBP, a metabolite of detoxification, and the second was a decrease in epoxidation, which is an activation process.


Formation of solid human mammary carcinoma in vitro. An established line of human breast carcinoma, MCF-7, when cultured within the artificial capillary system, formed solid masses of tissue. Acinar and ductular patterns developed, having the ultrastructural appearance of breast tissue that responds to exogenous hormones.


Induction in rats of paranasal sinus carcinomas with radioactive cerium chloride. A colloidal suspension of radioactive cerium stabilized in the form of hydroxide was inoculated into young Sprague-Dawley rats near the maxillary sinus. Well-differentiated epidermoid carcinomas of the paranasal sinus were obtained in 8 of 12 rats and in 8 of 9 animals that liver more then 200 days. Therefore, this istope was highly effectivein inducing tumors at the site of injection. The uniformity of the histologic type of tumors induced under these experimental conditions was remarkable.


Induction of pancreatic duct of carcinomas in the Syrian hamster with 2,6-dimethylnitrosomorpholine. Pancreatic duct carcinomas were induced in Syrian hamsters by intragastric administration of 2,6-dimethylnitrosomorpholine. As early as 30 weeks after treatment began, tumors were macroscopically visible in the pancreas; the maximum incidence of such tumors reached 71% by the end of the experiment. They were ductal in origin, and some metastasized to the lung. They were identified histologically as adenomas and carcinomas and developed in different parts of the organ without demonstrating any preferred localization.


Reduced growth rate of transplantable mammary adenocarcinoma in C3H mice fed eicosa-5,8,11,14-tetraynoic acid. Female 3-month-old C3H mice were given sc injections of 5-mg pieces of mammary adenocarcinoma and fed a linoleate-containing (15% corn oil) diet in the presence or absence of eicosa-5,8,11,14-tetraynoic acid (TYA), an inhibitor of prostaglandin synthesis. After 6 weeks, the weights of tumors of mice fed the TYA-free linoleate diet were three to five times greater than those of mice fed the TYA-containing linoleate diet. Dietary TYA caused a reduction in the levels of arachidonate and an elevation in the levels of linoleate in mammary tumors and livers. Aspirin, another known inhibitor of prostaglandin synthesis, when added to the linoleate diet, did not affect the tumor size or the composition of fatty acids in the tumors and livers. Thus we concluded that a) the growth of mammary tumors was not related to prostaglandin synthesis but was related to the availability of arachidonate, and b) TYA was an effective inhibitor for the conversion of linoleate to arachidonate.


United States-Japan cooperative cancer research program. The formalization of long-standing cooperative joint research efforts between Japanese and U.S. scientists has resulted in the development of specific areas of mutual program interest. The overall future objective of this program is to foster and encourage collaboration of mutual interest and benefit.


Massive fibroid.

Massive fibroid. This ten-year study of the massive fibroid at the Queen of Angels Hospital will reveal an average of 66 cases per year which could be classified as large and massive. Only about ten cases per year qualify as massive (four gestational months or larger). There were none considered giant size (25 lbs or more).The literature is replete with these, one of which (weighing 100.2 lbs) will be reported in detail. The mortality rate continues to be considerable in these (14.8 to 16.7 percent). In the smaller tumors, mortality is rare and morbidity is minimal.Bleeding, pain, and pressure symptoms, due to impingement on neighboring organs, are the principal symptoms. Sarcomatous change, fortunately, still remains quite rare.Treatment usually involves a pre-operative dilatation and curettage when bleeding is a problem, followed by total abdominal hysterectomy and bilateral salpingo-oophorectomy where indicated. Appendectomy is usually incidental. Anesthesia is usually spinal, if not otherwise contraindicated.Ultrasound is a new and refined diagnostic tool.


Medical aspects of sickle hemoglobin in military personnel. The Department of Defense (DOD) will soon issue a directive to test all incoming military personnel for the presence of hemoglobin S. The military testing program for hemoglobin S is an occupational medicine program. This report includes a discussion of armed services physical standards, a description of the Navy effort to evaluate an automated system for detection of hemoglobin S, and the proposed DOD directive.


Thyroglossal cyst in black patients.

Thyroglossal cyst in black patients. During the 16-year period, July 1, 1959 to June 30, 1975, inclusively, there were only 20 patients with thyroglossal cyst found at the Howard University Hospital (0.03 percent of routine surgicals). There were 12 males and eight females, all of whom were black. Anatomic and histopathologic variations of thyroglossal cysts will be emphasized, and the pitfalls in diagnosis discussed. A brief review of the literature will be presented.


Pitfalls in adolescent day treatment programming. The concept of the adolescent day treatment program offers many exciting treatment possibilities. This paper is an attempt to outline some of the potential problems or pitfalls which can occur in developing such a program. The author stresses the importance of having a program philosophy and indicates various aspects of the program which need careful planning and implementation.


Updating acute intermittent porphyria: a case of self-mutilation. Acute intermittent porphyria (AIP) as a precipitant of self-multilation in inmates, has not been documented. This case illustrates how AIP can go undiagnosed unless there is a high index of suspicion of this disease in the psychiatric patient, with persistent abdominal pains. This case illustrates the need to take seriously, the complaints of psychiatric patients, especially toxic-like symptoms, increasing paralysis, and abdominal pains. Perhaps acute intermittent prophyria has not been given the necessary attention in the literature which accounts for our failure to develop specific treatment methods.


Effect of feeding different carbohydrates to rats on low protein diets. The experimental results to be presented here do not demonstrate any significance among the means of the values obtained for the various carbohydrate diets, except for lactose. The results will suggest that the differences between the values for lactose and other diets are significant (PL0.05).No definite trend in the biological value (BV), the net protein utilization (NPU), and the true digestibility (TD) which can be attributed to the influence of the carbohydrates are shown by these results. The various dietary manipulations produce no results significantly different than the values for starch, the exception being lactose. The values for lactose are more than for other sugars.However, the results will show a relationship between a mixture of protein in the diet with the values of the individual protein sources if determined separately. Evidently, the values of each protein in any mixture can be predicted by measuring its TD in the mixture.


Predictive value of psychosocial profiles following aclte myocardial infarction. Psychosocial profiles were done prospectively on 54 males and three females in addition to hemodynamics and non-invasive studies; the average age at admission was 44 years. The mean study time for 45 survivors was two years and three months and was seven months for 12 non-survivors. Thirty potential psychosocial problems were investigated during coronary care unit stay, and at three months, six months, and one year. Four problems previously shown to have prognostic significance were re-examined. Depression and pessimism occurred more frequently in non-survivors (α = .05). Patients with maladaptive family relationships post-admission were more likely to die (66 percent), than those with adaptive relationships (15 percent) (α = .05). Patients with poor social adaptation prior to admission had significantly more psychosocial problems during follow-up (α = .05). However, this group had no more deaths than the total population. The type and number of psychosocial problems of survivors and non-survivors of acute myocardial infarction have predictive prognostic value.


Rush pin intramedullary fixation for fractures of the proximal humerus. We have treated almost 700 proximal humeral fractures, and selected from them a series of 16 cases which required operative intervention in the form of semiclosed reduction and internal fixation with the Rush pin because of the marked amount of displacement and risk on nonunion. We favor the 3/16 inch Rush pin for this operation. Fractures of the surgical neck comprise the vast majority of cases operated upon in this series. They are the easiest to reduce and fix by the Rush pin technique. Certain comminuted severely displaced fractures involving the anatomic neck of the humerus and/or greater and lesser tuberosities also lend themselves to this operation with comparable results to other methods of treatment. There have been no postoperative motalities and minimal morbidity. The operation can be performed rapidly and requires only a short period of immobilization and hospitalization.


Body fluid changes during hypertonic lactated saline solution therapy for burn shock. The body fluid changes of 12 burn patients treated with Hypertonic Lactate Saline solution (HLS group) were compared with 26 burn patients receiving isotonic therapy (iso Na group). 1) Total infusion volume during the first 48 hours postburn in the HLS group was only one-half to two-thirds of that in the iso Na group, although the sodium loads were nearly equal. 2) HLS therapy maintained sufficient functional extracellular fluid volume (f-ECFV) in the shock period and protected the excess increase of f-ECFV in the postresuscitative period, in contradistinction to the iso Na group. In this paper, some clinical problems such as a rapid shift of ECF water into the ICF space and hypernatremia following HLS administration are discussed.


The effect of antifibrinolytic therapy in experimental spinal cord trauma. The efficacy of antifibrinolytic therapy with epsilon aminocaproic acid (EACA) in experimental spinal cord injury is assessed by use of a standardized trauma model at both nonparaplegic and paraplegic trauma doses. Evaluation of neurologic parameters, cortical evoked response and histopathology of the injured spinal cord demonstrated that within the contusion range studied, EACA has no significant therapeutic effect. The ability of the animal to walk was the most reliable criterion indicative of functional recovery, and was closely paralleled by return of the cortical evoked response. Spinal cord cavitation in excess of 60% uniformly resulted in paraplegia. The data from this experiment show no evidence of a significant secondary injury occurring after spinal cord trauma that is amenable to posttraumatic antifibrinolytic therapy.


Percutaneous control of post-traumatic hepatic hemorrhage by gelfoam embolization. A case is reported in which a male laborer sustained severe hepatic lacerations after a tree fell across his abdomen. Despite intensive resuscitation and a laparotomy, bleeding continued from the liver and a second laparotomy was performed. When this also failed to control the hemorrhage a selective hepatic angiogram showed a false aneurysm in the right lobe of the liver with surrounding hematoma. This was successfully embolized with Gelfoam emboli. Bleeding ceased immediately with an uneventful recovery. The role of embolization in the management of hepatic trauma is discussed, especially in relation to surgical hepatic artery ligation.


Traumatic bilateral renal artery thrombosis. Although unilateral traumatic renal artery thrombosis may be more common than was once thought, bilateral traumatic renal artery thrombosis is rare, with only thirteen previously reported cases. Time is of the essence in diagnosing this condition, so that in the proper clinical setting one may be able to proceed quickly to arteriography. However, the prognosis at present still remains poor.


Vascular occlusion and stricture of the sigmoid colon secondary to trauma from a pneumatic hammer. A patient with stricture of the descending colon secondary to use of a pneumatic hammer is presented. Barium enema and angiographic studies suggested that the etiology was a traumatic vascular injury. Barium granulomata, found in the resected specimen, were presumably due to barium having entered the devitalized tissues during an air contrast study performed during an acute ischemic colitis. The mechanisms of traumatic strictures of the colon are discussed.


Steering wheel rupture of the pharyngoesophagus. A solitary injury. A unique case of a high posterior vertical linear laceration of the pharyngeosophagus secondary to steering wheel trauma without concomitant injury is presented. Diagnosis of this lesion is dependent upon frequently repeated physical examinations combined with appropriate laboratory and roentgenologic evaluations. Early institution of antibiotic therapy combined with early operative drainage and repair when feasible is advocated as the treatment of choice for this unusual entity.


Flow field-flow fractionation: new method for separating, purifying, and characterizing the diffusivity of viruses. The nature and theory of flow field-flow fractionation is described, and its potential applicability to virus-like particles is discussed. Different virus types are shown to be retained at different levels. Retention can be controlled by variation of the experimental parameters, in good agreement with theory. However, a mild adsorption effect is indicated and requires the development of alternate strategies for measuring diffusion coefficients. For Qbeta, our value agrees well within 10% of literature values; the values obtained for other viruses, using Abeta as an internal standard, are untested. Finally, it is demonstrated that flow field-flow fractionation can cleanly fractionate two viruses from one another and from an albumin impurity, that samples as large as several milligrams in size can be analyzed, and that the method has potential utility in the quantitative and qualitative analysis of virus systems.


In vitro translation with adenovirus polyribosomes. Polyribosomes isolated from adenovirus type 2 (Ad2)-infected HeLa cells late in productive infection can be used for translation in cell-free systems. At least eight viral polypeptides are synthesized, including the precursors to virion polypeptides VI and VII. Separation of polyribosomes by zonal rate centrifugation followed by translation in a cell-free system reveals a correlation between the sizes of the polyribosomes and the polypeptides synthesized. The cell-free extracts incorporate amino acid linearly for only 10 min and show little or no capacity to reinitiate protein synthesis. The elongation efficiency measured as the number of amino acids incorporated per ribosome in 20 min is low, ranging from 10 to 100. The maximum chain elongation rate is estimated to be 10 to 20 amino acids per min. The limited elongation has been used to assess the relative concentration of mRNA's engaged in translation.


Synthesis and processing of the precursor to the major core protein of adenovirus type 2. An isopycnic Metrizamide-detergent gradient system was developed in which the newly synthesized precursor (polypeptide P-VII) to the major core protein of adenovirus type 2 (polypeptide VII) was confined to a spectrum of complexes with densities equal to or higher than that of adenovirions. The majority of the newly synthesized P-VII was, at the beginning of the logarithmic period of virus production, present as an entity of protein density. This pool of P-VII was efficiently depleted. P-VII was also associated with high-molecular-weight structures of intermediate density, sharing some properties with empty capsids or incomplete particles. The transfer of P-VII from the intermediate-density region was not quantitative, and only particles of true virion density subsequently contained polypeptide VII. No structures equivalent to the core structure of disrupted virions or identical to incomplete particles were detected in this system. A temperature-dependent transition of radioactivity from polypeptide P-VII into polypeptide VII was also detectable after in vitro incubation of P-VII-containing complexes. Addition of Ad2-infected cell extracts was required for processing of complexes derived from regions of protein density, whereas P-VII was processed spontaneously upon incubation in complexes of virion density.


Biochemical, biophysical, and biological properties of densonucleosis virus (paravovirus). II. Two types of infectious virions. Among the infectious densonucleosis virus (DNV) particles, two types of virions, DNV-I and DNV-II, have been identified. The density of DNV-I was about 1.40 g/ml and depended on the pH, whereas the density of DNV-II was independent of the pH and was 1.44 g/ml. The sedimentation rates of these particles were 111S and 89S, respectively. The specific extinction coefficients and the absorption spectra, corrected for light scattering, were also different for these two types. The electrophoretic mobility of both types of virions was identical under the experimental conditions used. Both types of particles contained single-stranded DNA with similar physicochemical properties. The difference between these types of virions seemed to be due to a different protein content, DNV-II containing about 600,000 daltons less protein, and to a different quaternary structure as indicated by the change in the density of DNV-II, in the presence of divalent cations, without any change in the density of DNV-I.


Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA. The influenza virion transcriptase is capable of synthesizing in vitro complementary RNA (cRNA) that is similar in several characteristics to the cRNA synthesized in the infected cell, which is the viral mRNA. Most of the in vitro cRNA is large (approximately 2.5 X 10(5) to 10(6) daltons), similar in size to in vivo cRNA. The in vitro transcripts initiate in adenosine (A) or guanosine (G) at the 5' end, as also appears to be the case with in vivo cRNA (R.M. Krug et al., 1976). The in vitro transcripts contain covalently linked polyadenylate [poly(A)] sequences, which are longer and more heterogeneous than the poly(A) sequences found on in vivo cRNA. The synthesis in vitro of cRNA with these characteristics requires both the proper divalent cation, Mg2+, and a specific dinulceside monophosphage (DNMP), ApG or GpG. These DNMPs stimulate cRNA synthesis about 100-fold in the presence of Mg2+ and act as primers to initiate RNA chains, as demonstrated by the fact that the 5'-phosphorylated derivatives of these DNMP's, 32pApG or 32pGpG, are incroporated at the 5' end of the product RNA. The RNA synthesized in vitro differs from in vivo cRNA in that neither capping nor methylation of the in vitro transcripts has been detected. The virion does contain a methylase activity, as shown by its ability to methylate exogenous methyl-deficient Escherichia coli tRNA.


Effect of cyclophosphamide in vitro and on vaccinia virus replication in tissue culture. The effect of cyclophosphamide on the growth of Vero, BSC-1, and HeLa cells in monolayer cultures was studied. By using hemocytometer counts and tritiated thymidine uptake as indicators of growth, it was found that cyclophosphamide significantly interfered with the metabolism of Vero and BSC-1 cells when sustained in Leibovitz medium. Vero cells and HeLa cells grown in Eagle medium were not affected by exposure to cyclophosphamide. Vaccinia virus replication in Vero cell monolayer cultures incubated with cyclophosphamide was markedly augmented, and this enhanced growth was reflected by virus quantitation techniques and metabolic studies using tritiated thymidine uptake. No difference in the distribution of infectious particles was found when cyclophosphamide-treated and control infected cultures were compared. Pathways other than through hepatic enzymes appear available to activate cyclophosphamide in vitro. These effects are dependent on both the cell type and the medium in which the cells are grown. Cyclophosphamide can facilitate vaccinia virus replication in vitro through metabolic interactions at the cellular level. The precise mechanisms underlying this effect require further study.


Molecular weight of DNA from four entomopoxviruses determined by electron microscopy. DNA was isolated from entomopoxviruses infected Amsacta moorei and Euxoa auxiliaris (Lepidoptera), Goeldichironomus holoprasinus (Diptera), and Othnonius batesi (Coleoptera) and compared with vertebrate virus DNA (vaccinia). After incubation in Pronase, sodium lauryl sulfate, and deoxycholate, poxvirus preparations shadowed with platinum and palladium revealed subcore particles 45 to 60 nm in diameter. Continued incubation in Pronase resulted in the gradual release of DNA from the particles. Metal-shadowed DNA molecules were photographed in the electron microscope and measured, and the average molecular weights were calculated. Lepidopteran poxvirus DNA (135 X 10(6)) was approximately equal to vaccinia DNA (131.7 X 10(6)) in molecular weight. The molecular weight of dipteran and coleopteran poxvirus DNA (200 X 10(6) to 251 X 10(6)) was approximately 50% greater than vaccinia DNA. Based on the concentration of DNA and protein per virion, Amsacta entomopoxvirus contained 5.7 to 7.7% DNA.


Proteins of hepatitis B surface antigen. Purified 22-nm forms of hepatitis B surface antigen (Hbsag) representing the three major antigenic subtypes (adw, ayw, and adr) were analyzed for their constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No consistent difference in either the number or relative distributions of the polypeptides was observed for the various subtypes. Seven polypeptides were designated as P-1 through P-7 in order of their decreasing mobilities. By comparison with protein standards, their molecular weights were estimated as 23, 29.5, 36, 41.5, 53.5, 72, and 97 thousand. The P-1 and P-2 components represented the major polypeptides; P-2 and P-5 might by glycoproteins, based on their reaction with periodic acid-Shiff reagent. Each polypeptide contains cysteine residues. HBSAg was radiolabeled with 3H or 14C by reductive methylation or iodinated with 125I by the chloramine-T or lactoperoxidase procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled HBSAg yielded patterns identical to those obtained with protein stain. Comparison of HBSAg labeled by the chloramine-T and lactoperoxide procedures indicated that there was no distinction between internal or external components within the 22-nm structure.


Role of carbohydrate in biological functions of Friend murine leukemia virus gp71. Purified gp71 of Friend murine leukemia virus (FLV) can interfere with virus infection, absorb neutralizing antibody, and in the presence of group-specific anti-gp71 antibody, hemagglutinate sheep erythrocytes. Interference by FLV gp71 with several murine leukemia viruses (MuLV) was tested in the XC and S + L- assay systems. Treatment of gp71 with trypsin or Pronase eliminated its interfering capacity. However, treatment with neuraminidase or a mixture of glycosidase enzymes, which left the major serological properties of gp71 intact, did not reduce the interference potential of gp71 for FLV or AKR MuLV. The capacity of gp71 to absorb type- or group-specific virus-neutralizing antibodies was similarly affected by the various enzyme treatments. In contrast, indirect hemagglutination by gp71 was abolished not only by proteases but also by treatment with glycosidase enzymes, although neuraminidase had no effect. Preliminary data indicate that infectivity of FLV or xenotropic MuLV was not affected by short treatment with glycosidase enzymes.


DNA contained by two densonucleosis viruses. The DNA contained by particles of densonucleosis viruses 1 and 2 were analyzed within the particle, and properties of DNA extracted from these particles were determined. The DNA appears to exist as a single-stranded molecule with limited secondary structure within particles, as assessed by spectral changes induced by formaldehyde, melting profiles, and circular dichroism studies. The single-stranded DNA had an apparent molecular weight of 1.9 X 10(6) to 2.2 X 10(6) as assessed by differences in the molecular weight of virus particles and top component and percentage of nucleic acid. DNA extracted from virus particles in low-salt buffers possessed properties typical of a single-stranded molecule. Double-stranded DNA could be extracted from virus particles under appropriate high salt and elevated temperature. The linear double-stranded DNA extracted from both viruses had a molecular weight of about 3.9 X 10(6) to 4.1 ZX 10(6) determined by neutral sedimentation and electron microscopy and an equivalent genome size determined by reassociation kinetics. About 87% of the DNA was homologous between the two viruses.


Polyamines contained by two densonucleosis viruses. Densonucleosis viruses 1 and 2 both contain the three polyamines putrescine, spermidine, and spermine in complete virus particles but not in their respective top components. The polyamines, with spermidine predominant, comprise 1.41% of the virus particle by weight, which is sufficient to neutralize 26% of the single-stranded DNA contained within the particles.


Polypeptides of respiratory syncytial virus. Radiolabeled respiratory syncytial virus was purified from medium that had been harvested from infected HeLa cell monolayers before it contained much cellular debris. After isopycnic centrifugation in linear gradients prepared with sucrose dissolved in Hanks balanced salt solution, almost all the infectivity and most of the radioactivity were recovered in a single band with density from 1.16 to 1.23 g/cm3 and a peak at 1.2 g/cm3. Analysis by polyacrylamide gel electrophoresis resolved the purified virus into seven polypeptides of approximate molecular weights 20,000 to 80,000, of which the two largest and the smallest proved to by glycoproteins.


Complementation analysis of measles virus temperature-sensitive mutants. Two sets of independently isolated measles virus temperature-sensitive mutants were quantitatively tested for complementation. Analysis of the nine possible combinations of representative mutants indicated that only one pair of mutants is noncomplementing. Thus, the measles virus mutants studied to date define five complementation groups.


DNA synthesized in the hepatitis B Dane particle DNA polymerase reaction. Radioactive DNA was prepared in extensive (4 h) Dane particle DNA polymerase reactions. In different experiments the amount of new DNA, determined by the amount of nucleotide incorporation into an acid-insoluble form, was between 29 and 45% of the total circular DNA isolated from Dane particle preparations after the reaction. DNA reassociation kinetics were used to determine the complexity of the newly synthesized DNA. In different experiments COt1/2 values, corresponding to between 625 and 1,250 nucleotide pairs, were obtained for the radioactive Dane particle DNA. These results suggest that a unique region (or regions), corresponsing to approximately one-fourth to one-half of the circular Dane particle DNA template, was copied one time during the reaction. DNA and RNA extracted from hepatitis B virus-infected liver but not from uninfected liver accelerated the rate of reassociation of radioactive DNA from Dane particles. These Dane particle DNA base sequences were found in alkali-stable, rapidly sedimenting DNA from infected liver as well as in DNA sedimenting at a rate similar to the DNA extracted from Dane particles. These findings are consistent with Dane particle DNA being hepatitis B virus DNA that is integrated into high-molecular-weight cellular DNA and transcribed into RNA in infected liver.


Synthesis of mRNA guanylyltransferase and mRNA methyltransferases in cells infected with vaccinia virus. Guanylyltransferase and methyltransferases that modify the 5'-terminals of viral mRNA's to form the structures m7G(5')pppAm- and m7G(5')pppGm- appear to be synthesized afte- vaccinia virus infection of HeLa cells. Elevations in these enzyme activities were detected within 1 h after virus inoculation and increased 15- to 30-fold by 4 to 10 h. Increases in the guanylyl- and methyltransferase activities were prevented by cycloheximide, an inhibitor of protein synthesis, but not by cytosine arabinoside, an inhibitor of DNA synthesis. The latter results suggest that the mRNA guanylyl- and methyltransferases are "early" or prereplicative viral gene products. The guanylyltransferase and two methyltransferases, a guanine-7-methyltransferase and nucleoside-2'-methyltransferase, were isolated by column chromatography from infected cell extracts and found to have properties similar or identical to those of the corresponding enzyme previously isolated from vaccinia virus cores. In contrast, enzymes with these properties could not be isolated from uninfected cells.


In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: solubilization of membrane-bound replicase and partial purification. A fraction containing membrane-bound tobacco mosaic virus RNA replicase was isolated form tobacco mosaic virus-infected tobacco callus cultures. The replicase activity reached a maximum 60 h after inoculation and then declined. The enzyme activity was insensitive to actinomycin D and DNase. The corresponding fraction from healthy callus contained essentially no activity. The viral RNA synthesis in vitro proceeded linearly for 30 min and required the four nucleotide triphosphates and Mg2+ ions. Mn2+ was a poor substitute for Mg2+. During RNA synthesis the product was at least 70% resistant to RNase in 2X SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digested by RNase in 0.1X SSC. Analysis of the product by polns) that appeared to be replicative form and a partially RNase-resistant structure similar to replicative intermediate form. Washing the membrane-bound replicase with Mg2+-deficient buffer solubilized enzyme. The solubulized enzyme was further purified by DEAE-Sephadex column chromatography. The DEAE-purified enzyme was nearly completely dependent upon tobacco mosaic virus RNA for activity. Analysis of the product on a sucrose gradient revealed a double-stranded RNA with sedimentation of 16S and smaller heterogeneous RNase-sensitive products.


Functions of gene C and gene D products of bacteriophage phi X 174. Phage-related materials existing in cells infected with various mutants of bacteriophage phi chi 174 were investigated. A novel species of replicative-form (RF) DNA was found in cells infected with a phage mutant of gene B, C, D, F, or G. This species, called RFI, sedimented at a position between RFI and RFII in a neutral sucrose gradient. It was converted to RFI upon denaturation in alkali, denaturation in formamide and subsequent renaturation, or RNase treatment at low ionic strength. In cells infected with a phage mutant of gene C, RFI was derived from pulse-labeled RFII after a short chase. TLLS INFECTED WITH A MUTANT OF GENE B, D, or F. A possible function of the C gene product of phi chi 174 could be to prevent the conversion of RFII to RFI, thereby maintaining the availability of RFII to act as the template for single-stranded viral DNA synthesis. A protein complex containing no DNA, which sedimented with an S value of 108 in a sucrose gradient and contained virion proteins F, G, and H, and nonvirion protein D, was found in cells infected with the gene C mutant. A possible function of protein D was considered as a scaffolding protein for assembly of phage structural proteins.


Use of specific radioactive probes to study transcription and replication of the influenza virus genome. Specific radioactive probes have been obtained for both influenza virion RNA (vRNA) and for its complement (complementary RNA or cRNA): 32P-labeled complementary DNA (cDNA) synthesized with the avian sarcoma virus reverse transcriptase, and [125I]vRNA, respectively. From the kinetics of annealing of these two probes to RNA from canine kidney cells infected with the WSN strain of influenza virus, we have determined the average number of cRNA and vRNA sequences in the nucleus and cytoplasm as a function of time after infection. Immediately after infection, a small amount of vRNA is detected, presumably from the inoculum virus. As expected, the amount of cRNA is insignificant. During the first 1.75 h of infection, the most significant increase observed is in cRNA sequences. Most of these cRNA sequences are found in the cytoplasm, but a significant amount (30%) is found in the nucleus. During this time, a small but significant increase in vRNA is also detected in the nucleus and cytoplasm. From 1.75 to 2.75 h, the absolute amounts of both cRNA and vRNA increase, predominantly in the cytoplasm, with cRNA remaining as the majority species. Subsequently, the amount of vRNA increases with respect to cRNA and becomes the majority species. At 3.75 h, 95% of both cRNA and vRNA are found in the cytoplasm. Addition of actinomycin D at 1.75 h completely suppresses the subsequent ninefold increase in cRNA and does not have a significant effect on the subsequent 14-fold increase in cytoplasmic vRNA. This assay is also able to detect the cRNA produced as a result of primary transcription, operationally defined as the cRNA produced in the presence of 100 mug of cycloheximide per ml added at zero time of infection. Increases in cRNA in the presence of cycloheximide are detectable in both the nucleus and the cytoplasm. Addition of actinomycin D as well as cycloheximide at zero time completely suppresses the appearance of cRNA in the cytoplasm, whereas a large fraction (50%) of the increase in nuclear cRNA still occurs.


Free and membrane-bound polyribosomes in BHK cells infected with Sindbis virus. The data presented in the paper demonstrate that in BHK cells infected with Sindbis virus virtually all the 42S mRNA not in nucleocapsid is associated with free polyribosomes, whereas the 26S mRNA is distributed between free and membrane-bound polyribosomes. We suggest that the 26S RNA polyribosomes are bound to the membranes through the nascent chains of the B1 protein and that a large percentage of 26S RNA polyribosomes free in the cytoplasm may be due to the small amount of rough endoplasmic reticulum in BHK cells. In addition, we found that intracellular nucleocapsid is in the nonmembrane fraction of the cytoplasm of infected cells.


Suppressors of gene 32 am mutants that specifically overproduce P32 (unwinding protein) in bacteriophage T4. A gene 32 amber (am) mutant, amNG364, fails to grow on Escherichia coli Su3+ high temperatures, suggesting that the tyrosine residue inserted at the am codon by Su3+ leads to a temperature-sensitive gene 32 protein (P32). By plating amNG364 on E. coli Su3+ 45 degrees C, several pseudorevertants were found that proved to contain a suppressor (su) mutant in addition to the original am mutation. Crosses of two of these amNG364su strains to am+ phage indicated that the suppressors themselves are in or close to gene 32. Phage strains carrying either of the two su mutations, without amNG364, grew normally. When cells were infected by these su mutants and the proteins produced were examined by sodium dodecyl sulfate-gel electrophroesis, specific overproduction of P32 was found. Maximum overproduction compared to am+ phage was 6.6-fold for one su mutant and 2.4-fold for the other. Other proteins were produced in normal amounts and in normal time sequence. When amNG364su phage were allowed to infect E. coli S/6/5(Su-), the gene 32 am fragments produced were present at the same derepressed levels as in an infection by amNG364 without a suppressor. The suppressor mutations are interpreted as causing derepression of P32 by altering sites in this autogenously regulated protein involved in template recognition. Previously, specific derepression of gene 32 had only been shown using gene 32 conditional lethal mutants grown under restrictive conditions. We have shown that P32 can also be derepressed under permissive conditions, indicating that loss of P32 function is not necessary for specific derepression.


Induction and biological properties of defective interfering particles of rabies virus. A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.


Structural components of influenza C virions. The genome RNA species of influenza type C virions were analyzed by polyacrylamide gel electrophoresis. The pattern obtained was found to resemble those of other influenza viruses. Six RNA species were resolved, with estimated sizes ranging from 0.37 X 10(6) to 1.25 X 10(6) daltons. The internal ribonucleoproteins of influenza C virions were found to sediment heterogeneously in glycerol velocity gradients as demonstrated previously with influenza A/WSN virus. The ribonucleoproteins possessed diameters of 12 to 15 nm, with lengths ranging from 30 to 100 nm. Of the three major virion polypeptides (molecular weights, 88,000, 66,000, and 26,000), only the largest is glycosylated. Similar polypeptide species were present in influenza C virions of five different strains. All three major proteins of influenza C virions possess electrophoretic mobilities distinguishable from those of the major polypeptides of influenza A/WSN. The 66,000-dalton protein is associated with the ribonucleoprotein components. Two additional glycosylated polypeptides, with estimated molecular weights of 65,000 and 30,000, were detected in virions grown in embryonated eggs, but not in virus particles obtained from chicken embryo fibroblasts.


Structure of hepatitis B Dane particle DNA before and after the Dane particle DNA polymerase reaction. DNA isolated from the hepatitis B antigen form known as the Dane particle was examined by electron microscopy before and after the endogenous Dane particle DNA polymerase reaction. The most frequently occurring form was an untwisted circular double-stranded DNA molecule approximately 1 mum in length. Less frequently occurring forms included circular DNA of approximately unit length and having one or more small single-stranded regions, similar circular molecules with one or more tails either shorter or longer than 1 mum in length, and very small circular molecules with tails. There was no increase in frequency or length of tails after a DNA polymerase reaction, suggesting that tails were not formed during this reaction. The mean length of circular molecules increased by 23% when DNA was spread in formamide compared with aqueous spreading, suggesting that single-stranded regions are present in most of the molecules. The mean length of circular molecules obtained from aqueous spreading increased by 27% after a Dane particle DNA polymerase reaction. This indicates that single-stranded regions were converted to double-stranded DNA during the reaction.


Identification of the site of interruption in relaxed circles producing during bacteriophage lambda DNA circle replication. The DNA that accumulates in the lambda infection restricted to the early (circular) stage of replication consists of approximately two-thirds covalently closed circles and one-third relaxed circles bearing a single interruption in either strand of the duplex. The latter molecules are presumed to be a unique class in that the interruption is not repairable by DNA polymerase and ligase. Preferential radioisotopic labeling of the region immediately adjacent to the interruption, followed by hybridization to sheared fragments of the lambda chromosome with varying guanine plus cytosine content, suggested that the nick resides at the position of the mature molecular ends of the lambda chromosome. Digestion of the labeled molecules with restriction enzymes and reconstruction experiments in which Hershey circles were generated by annealing of interrupted strands isolated from the relaxed circles support this interpretation. The results indicate that the relaxed circles consist of a population containing one interruption in either of the two strands of the duplex jointly representing the two "nicks" contained in Hershey circles (in which the cohesive ends are annealed). These molecules could result from the inability of the maturation function to make the required staggered endonucleolytic cuts when the DNA substrate is a monomeric circle rather than a multimeric linear molecule. Alternatively, this interruption could be the result of an endonucleolytic cutting event critical to DNA replication.


Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis. The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).


Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy. Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.