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The effect of atenolol and reserpine on incidence of strokes, coronary heart disease (CHD), cardiovascular disease (CVD), and mortality was assessed in 4736 persons aged 60 years and older with isolated systolic hypertension. Participants were randomized to either chlorthalidone (2371), with step-up to atenolol, or reserpine if needed, or placebo (2365). The average baseline SBP/DBP was 170/77 mm Hg. In the active treatment group, step 1, dose 1 was chlorthalidone, 12.5 mg/day; dose 2 was 25 mg/day. For step 2, dose 1 was atenolol 25 mg/day (or reserpine 0.05 mg/day if atenolol was contraindicated); dose 2 was 50 mg/day (reserpine, 0.10 mg/day). During 4.5 years average follow-up, 32% (757) of the active treatment group were on atenolol, with an average exposure of two years and 8% (193) were on reserpine with an average exposure of 1.7 years. Overall there were 96 strokes, 140 CHD events and 289 CVD events among the 2365 active group participants. Using time-dependent lifetable regression with adjustment for several variables, the addition of either atenolol or reserpine to chlorthalidone did not substantially alter the risk ratios for chlorthalidone alone. The relative risk for CHD events for atenolol versus no atenolol was 1.04 (95% confidence interval: 0.58, 1.86) and for reserpine versus no reserpine was 0.93 (95% confidence interval: 0.29, 2.96). The relative risk for atenolol were 0.84 (95% confidence interval: 0.54, 1.30) for death, 1.34 (95% confidence interval: 0.80, 2.28) for stroke, and 1.07 (95% confidence interval: 0.71, 1.61) for CVD. For reserpine, the corresponding relative risks and confidence intervals were 0.65 (0.26, 1.59) for death, 0.27 (0.04, 2.26) for stroke, and 0.55 (0.20, 1.49) for CVD. Thus, the beneficial effects in several outcomes in Systolic Hypertension in the Elderly Program (SHEP) were due to the treatment regimen of lowering blood pressure based on low-dose chlorthalidone (plus atenolol or reserpine as required to meet blood pressure criteria). Additional (independent) benefits attributable to atenolol or to reserpine were not identified. However, a greater number of patients might have been necessary to adequately evaluate potential differential effects of these drugs, especially for reserpine.

Four hundred fifty Escherichia coli strains of 45 O serogroups and subgroups and 112 serotypes were studied to determine their patterns of adherence to HeLa cells. Adherence was exhibited by strains of 17 O serogroups and subgroups, but within these groups more than one adherence pattern was frequently observed. However, within each serotype, the adherence pattern was highly consistent. Localized adherence (LA) was observed much more frequently in serotypes that we considered to be enteropathogenic E. coli serotypes (93%) than in other serotypes (14%), whereas diffuse adherence (DA) occurred predominantly among nonenteropathogenic E. coli strains. Determination of biochemical characteristics showed that within O serogroups, nonmotile strains tended to have the same behavior as motile strains with the LA adherence pattern, suggesting that they were derived from these motile strains. LA and non-LA strains of the same serotype differed biochemically. LA appears to be a property of most E. coli commonly considered to be enteropathogenic and should assist attempts to determine which E. coli are enteropathogenic and to elucidate their pathogenic mechanisms.

The relation between coffee consumption and serum cholesterol level was investigated in a group of 9,043 hypertensive adults who were in the Hypertension Detection and Follow-up Program. In this study, men and women aged 30-69 years at baseline (1973-1974) had their serum cholesterol level measured at the two-year examination (1975-1976). Information about coffee, tea, and cola consumption was also obtained at that time from a food frequency questionnaire. The relation of coffee consumption and serum cholesterol level with potentially confounding variables including age, race, sex, diuretic status, diastolic blood pressure, cigarette smoking, relative weight, physical activity, stress, and education level was examined. When these variables were entered into a multiple regression equation, a positive association with coffee consumption and serum cholesterol level (p less than 0.05) was present. There was no significant relation between serum cholesterol level and consumption of tea, cola, or decaffeinated coffee--the other major contributors of caffeine to the diet--or total caffeine intake. This study indicates a significant positive relation between coffee consumption and serum cholesterol level.

Infection of mice with Moloney murine leukemia virus (M-MuLV) as well as with a nonpathogenic variant, Mo+PyF101 M-MuLV, was studied. Mo+PyF101 M-MuLV differs from wild-type M-MuLV by the addition of enhancer sequences from polyomavirus in the long terminal repeat. Previous experiments indicated that Mo+PyF101 establishes infection in animals, even though it does not induce disease. In vivo infection studies with particular attention to the thymus were performed, since the thymus is the target organ for M-MuLV leukemogenesis. Mice inoculated at birth with wild-type M-MuLV developed maximal levels of thymic infection by 2 to 3 weeks. Animals inoculated with Mo+PyF101 M-MuLV showed considerably less thymic infection at early times (2 to 4 weeks); nevertheless, by 5 to 6 weeks infection equivalent to wild-type M-MuLV-inoculated animals developed. Therefore the nonpathogenicity of Mo+PyF101 M-MuLV did not simply reflect a lack of thymotropism. Furthermore, thymic infection by itself may not be sufficient to induce leukemia. The relative deficit of Mo+PyF101 M-MuLV thymic infection at early versus late times did not reflect a change in the nature of the cells in the thymus, since in vitro infection of primary thymocytes from 2- and 6-week-old animals was equally efficient. One possible explanation is that infected thymocytes normally arise from progenitor cells which were infected in the bone marrow or spleen, and the cells restricted for Mo+PyF101 M-MuLV are located in those organs. Comparison of wild-type and Mo+PyF101 M-MuLV also allowed identification of important preleukemic changes in the thymus of wild-type M-MuLV-inoculated mice. Flow cytometry with monoclonal antibodies specific for thymocyte subpopulations was used. Staining of cells for Thy-1 or Thy-1.2 antigens indicated a shift toward low or negative cells. A concomitant increase in cells positive for antigen Pgp-1 was also observed. This is consistent with an increase in the relative frequency of immature blastlike cells. Importantly, thymuses from mice inoculated with Mo+PyF101 M-MuLV did not show these shifts in thymocyte subpopulations.

A Moloney murine leukemia virus (M-MuLV) recombinant carrying the v-src gene of avian sarcoma virus was generated by the introduction of a cloned portion of v-src from Schmidt-Ruppin A avian sarcoma virus into a molecular clone of M-MuLV provirus at the recombinant DNA level. The v-src sequences (lacking a portion of the 5' end of v-src) were inserted into the p30 region of the M-MulV gag gene so that M-MuLV gag and v-src were in the same reading frame. Transfection of this chimeric clone, pMLV(src), into NIH 3T3 cells which were constitutively producing M-MuLV gag and pol protein resulted in the formation of foci of transformed cells. Infectious and transforming virus could be recovered from the transformed cells. This virus was designated M-MuLV(src). M-MuLV(src)-transformed cells contained two novel proteins of 78 and 90 kilodaltons. The 78-kilodalton protein, p78gag-src, contained both gag and src determinants, exhibited kinase activity in an immune kinase assay, and is probably a fusion of Pr65gag and src. The 90-kilodalton protein, which is of the appropriate size to be the gPr80gag fused to src, contained gag determinants as well as a V8 protease cleavage fragment typical of the carboxy terminus of avian sarcoma virus pp60src. However, it could not be immunoprecipitated with an anti-v-src serum. M-MuLV(src)-transformed cells showed elevated levels of intracellular phosphotyrosine in proteins, although the elevation was intermediate compared with cells transformed with wild-type v-src. M-MuLV and amphotropic murine leukemia virus pseudotypes of M-MuLV(src) were inoculated into newborn NIH Swiss mice. Inoculated mice developed solid tumors at the site of inoculation after 3 to 6 weeks, with most animals dying by 14 weeks. Histopathological analysis indicated that the solid tumors were mesenchymally derived fibrosarcomas that were both invasive and metastatic.

Two large, independently obtained collections of vibrio cholerae non O1, containing 59 and 67 reference strains, respectively, were compared serologically in four laboratories. Twenty strains in each collection were considered identical, and an additional 16 strains in each collection were probably identical. Twenty-eight unique strains were identified, and inconclusive results were obtained with 25 strains. Nine strains were not considered V. cholerae in at least one testing laboratory. Of these, five showed insufficient correspondence to V. cholerae O1 by DNA hybridization to be considered V. cholerae.

Antibiotic disk susceptibility tests were done on 220 strains of Escherichia coli belonging to serotypes reported in the literature to be associated with the production of enterotoxin. A total of 128 (58%) were resistant to one or more antibiotics, sulfa drugs, or chemotherapeutic agents. An analysis of these strains revealed primary, secondary, and tertiary drug resistance patterns that indicated a selective pattern in the formation of multiple drug resistance in E. coli. Resistances to certain antibiotics were more likely to occur in pairs and triads (secondary resistance patterns) that were often combined or coexisted in a single strain of E. coli to produce tertiary drug resistance patterns, conferring drug resistance to five or six different antibiotics. Among enterotoxin-associated serotypes, single and multiple drug resistance was less frequently associated with enterotoxin-produced strains than with strains from the same serotype that were not enterotoxigenic. Within the enterotoxigenic E. coli, single and multiple resistance to antibiotics was more frequent in strains producing only heat-stable enterotoxin (ST) than in strains producing only heat-labile enterotoxin (LT) or both. The number of resistances to different antibiotics per resistant strain averaged approximately 1.4 for LT plus ST or LT strains, and 3.9 for ST strains and nonenterotoxigenic strains. Phenotypic characterization of 170 strains for four usually plasmid-mediated characteristics showed that the number of antibiotics to which a strain was directly resistant varied with the type and number of plasmid-mediated characteristics present.

We screened 223 strains of Escherichia coli belonging to serotypes previously associated with the production of enterotoxin for hemolytic activity, using horse erythrocytes in liquid and in agar media. Thirty-eight were hemolytic. They belonged to nine different serotypes; most (65.8%) belonged to one serotype, O6: H-. Additionally, all 38 strains were specifically assayed for a filterable, heat-labile hemolytic activity previously associated with a hemolysin plasmid. A comparison of hemolytic activity and enterotoxicity showed that none of 32 strains hemolytic in both media was enterotoxigenic; 28 of the 32 expressed heat-labile hemolytic activity. Four of the six strains hemolytic in only one of the media were enterotoxigenic; none of these six expressed heat-labile hemolytic activity. Of 223 strains, 176 that were of human origin and isolated in the United States were further assayed for three traditionally plasmid-mediated characteristics: heat-labile enterotoxin, heat-stable enterotoxin, and colonization factors. The interrelationships of these characteristics, including hemolytic activity, may reflect varying degrees of plasmid compatibility.

Escherichia coli serotype O157:H7 has been isolated from outbreaks and sporadic cases of hemorrhagic colitis. There is convincing evidence that it can cause this diarrheal disease. Because of the interest in hemorrhagic colitis, it has become desirable to detect this particular strain in human feces, which usually contains many other strains of E. coli. Two characteristics of the incriminated E. coli O157:H7 strain have made its isolation and identification easier. It does not ferment D-sorbitol rapidly, in contrast to about 95% of other E. coli strains. In addition, the strain has H antigen 7, but only about 10% of other E. coli strains have this particular antigen. To screen for E. coli O157:H7 we devised H7 antiserum-sorbitol fermentation medium (18 g of enteric fermentation base, 10 g of D-sorbitol, 4 g of agar, 10 ml of Andrade indicator, 989 ml of water; all ingredients were mixed, autoclaved, and cooled; 1 ml of E. coli H7 antiserum was then added). Colonies to be screened were inoculated into this medium. Strains of E. coli O157:H7 gave a characteristic pattern; they did not ferment sorbitol and were immobilized in the semisolid medium because of the reaction of their flagella with the flagella antiserum. Almost all other strains of E. coli gave a different pattern; they fermented sorbitol or were not immobilized by the H7 serum or both. Strains which were presumptive positives (sorbitol negative, H7 positive) were then tested in E. coli O157 serum by slide or tube agglutination. The number of strains which were presumptive positive by H7-sorbitol medium but then were not found to be O157 was less than 1%. A second approach has been helpful in deciding which colonies to screen in H7-sorbitol medium. MacConkey-sorbitol agar (22.2 g MacConkey agar base [which contains no sugar], 10 g of D-sorbitol, 1,000 ml of water) was designed as a plating medium. Stools were plated on MacConkey agar to estimate the number of E. coli colonies and also plated on MacConkey-sorbitol agar to estimate the number of sorbitol-negative colonies of E. coli. These two approaches have proved useful for isolating and identifying E. coli O157:H7 form human feces and from feces of animals infected in the laboratory with this strain. The results suggest that media may be formulated in a similar fashion for detecting other specific strains of E. coli.

Two outbreaks of hemorrhagic colitis, a newly recognized syndrome characterized by bloody diarrhea, severe abdominal pain, and little or no fever, occurred in 1982. No previously recognized pathogens were recovered from stool specimens from persons in either outbreak. However, a rare E. coli serotype, O157:H7, was isolated from 9 of 20 cases and from no controls. It was also recovered from a meat patty from the implicated lot eaten by persons in one outbreak. No recovery of this organism was made from stools collected 7 or more days after onset of illness; whereas 9 of 12 culture-positive stools had been collected within 4 days of onset of illness. The isolate was not invasive or toxigenic by standard tests, and all strains has a unique biotype. Plasmid profile analysis indicates that all outbreak-associated E. coli O157:H7 isolates are closely related. These results suggest that E. coli O157:H7 was the causative agent of illness in the two outbreaks.

DNA relatedness was used to define the biochemical boundaries of Escherichia coli. A large number of biochemically atypical strains were shown to belong to biogroups of E. coli. These included strains negative in reactions for indole, all three decarboxylases, D-mannitol, lactose, or methyl red and strains positive in reactions for H2S, urea, citrate, KCN, adonitol, myo-inositol, or phenylalanine deaminase. Frequency and source data are presented for these atypical E. coli biogroups. One group of KCN-positive, cellobiose-positive, yellow-pigmented strains was 84 to 91% interrelated but only 35 to 45% related to E. coli. The name Escherichia hermannii sp. nov. is proposed for this group of organisms that was formerly called Enteric Group 11 by the Enteric Section, Centers for Disease Control, Atlanta, GA. Twenty-nine strains of E. hermannii have been isolated in the United States from a variety of clinical sources, principally wounds, sputum, and stools. Three additional strains were isolated from food. E. hermannii strains are gram-negative, oxidase-negative, fermentative, motile rods. In addition to yellow pigment and positive KCN and cellobiose tests, the biochemical reactions characteristic of 32 strains of E. hermannii were as follows: gas from D-glucose, acid from D-glucose, maltose, D-xylose, L-arabinose, L-rhamnose, and D-mannitol; no acid from adonitol or inositol; variable acid production from lactose and sucrose; positive tests for indole, methyl red, and mucate; negative tests for Voges-Proskauer. Simmons citrate, H2S, urea, phenylalanine deaminase, and gelatin hydrolysis; negative or delayed test for L-lysine decarboxylase and negative test for L-arginine dihydrolase; and positive test for ornithine decarboxylase. E. hermannii strains were resistant to penicillin, ampicillin, and carbenicillin and sensitive to other commonly used antibiotics. Wounds account for almost 50% of human isolates of E. hermannii, followed by sputum or lung isolates (ca. 25%) and stool isolates (20%).

Forty-five strains of ureolytic Escherichia coli of human origin, isolated in the United States between 1956 and 1977, were characterized by geographical distribution, site of infection, serotype, resistance to antibiotics, and biochemical reactions. All strains were studied for the ability to generate clones of nonureolytic E. coli (segregants), and a subset of these were selected for plasmid analysis and a variety of bacterial matings. There did not appear to be a common geographical distribution, serotype, antibiogram, or other aberrant biochemical reactions other than the hydrolysis of urea among these strains. The predominance of urinary tract isolates (46.7% total) may reflect a relationship between urea hydrolysis and pathogenesis at this site. Ten of the strains (22.2%) did segregate nonureolytic E. coli colonies, and all possessed at least one common plasmid species with a molecular weight of about 65 X 10(6). Only strain 1138-77 serotype O16:H6 conjugally transfered the ability to hydrolyze urea, ferment sucrose, and resist inhibition by sulfadiazide simultaneously. The resulting, recombination-deficient E. coli K-12 tranconjugant was found to possess a plasmid with a molecular weight of about 80 X 10(6) to 90 X 10(6).

Biochemically atypical strains classified as Vibrio cholerae were characterized by biochemical reactions, serology, antibiotic susceptibility testing, and deoxyribonucleic acid relatedness. Strains with the following atypical reactions were shown to be V. cholerae: mannose negative, mannitol negative, lysine decarboxylase negative, no growth in the presence of 5% NaCl, salicin and cellobiose positive. Sucrose-negative strains were shown to constitute a new species, Vibrio mimicus, whose type strain is 1721-77 (ATCC 33653). In addition to its negative sucrose reaction, V mimicus was differentiated from V. cholerae by its negative Voges-Proskauer, corn oil, and Jordan tartrate reactions and by its sensitivity to polymyxin. V. mimicus was isolated from shellfish and water, as well as from human diarrheal stools and ear infections. Most strains were typable with antisera against V. cholerae. Strains from three serogroups produced either a heat-labile or a heat-stable enterotoxin.

No new serotypes have been added to the Shigella schema since 1958, although several provisional serotypes have been described. We conducted biochemical and serological studies on three provisional Shigella boydii serotypes. Four strains of serotype 2710-54 from four widely separated countries, 7 strains of serotype 3615-53 from three different countries, and 31 strains of serotype 1344-78 (E10163) from six different countries were included. Reactions of all three serotypes were consistent with those of S. boydii. On the basis of these results and other published research, we propose that these three provisional serotypes be admitted to the Shigella schema as S. boydii 16, 17, and 18.

Escherichia fergusonii (formerly known as Enteric Group 10) and Enterobacter taylorae (formerly known as Enteric Group 19) are proposed as new species in the family Enterobacteriaceae. By DNA hybridization (32P, 60 degrees C, hydroxyapatite), strains of E. fergusonii were 90 to 97% related to the type strain (holotype) ATCC 35469. They were most closely related to Escherichia coli and more distantly related to species in other genera. E. fergusonii strains are positive for indole production, methyl red, lysine decarboxylase, ornithine decarboxylase, and motility. They ferment D-glucose with gas production and also ferment adonitol, L-arabinose, L-rhamnose, maltose, D-xylose, trehalose, cellobiose, and D-arabitol. They are negative for Voges-Proskauer, citrate utilization (17% positive), urea hydrolysis, phenylalanine deamination, arginine dihydrolase, growth in KCN, and fermentation of lactose, sucrose, myo-inositol, D-sorbitol, raffinose, and alpha-methyl-D-glucoside. By DNA hybridization (32P, 60 degrees C, hydroxyapatite), strains of E. taylorae were 84 to 95% related to the type strain (holotype) ATCC 35317. Their nearest relative was E. cloacae, to which they were 61% related. Other named species were more distantly related. Strains of E. taylorae are positive for Voges-Proskauer, citrate utilization, arginine dihydrolase, ornithine decarboxylase, motility, growth in KCN medium, and malonate utilization. They ferment D-glucose with gas production and also ferment D-mannitol, L-arabinose, L-rhamnose, maltose, D-xylose, trehalose, and cellobiose. They are negative for indole production, methyl red, H2S production on triple sugar-iron agar, urea hydrolysis, phenylalanine deamination, lysine decarboxylase, gelatin hydrolysis, and fermentation of adonitol, i-inositol, D-sorbitol, and raffinose. Both new species occur in human clinical specimens. Two strains of E. fergusonii were isolated from blood. Five stains of E. taylorae were isolated from blood, and one was from spinal fluid. These blood and spinal fluid isolates suggest possible clinical significance, but this point requires further study.

In 1972 there were only 11 genera and 26 species in the family Enterobacteriaceae. Today there are 22 genera, 69 species, and 29 biogroups or Enteric Groups. This paper is a review of all of the new organisms. It has a series of differential charts to assist in identification and a large chart with the reactions of 98 different organisms for 47 tests often used in identification. A simplified version of this chart gives the most common species and tests most often used for identification. The sources of the new organisms are listed, and their role in human disease is discussed. Fourteen new groups of Enterobacteriaceae are described for the first time. These new groups are biochemically distinct from previously described species, biogroups, and Enteric Groups of Enterobacteriaceae. The new groups are Citrobacter amalonaticus biogroup 1, Klebsiella group 47 (indole positive, ornithine positive), Serratia marcescens biogroup 1, and unclassified Enteric Groups 17, 45, 57, 58, 59, 60, 63, 64, 68, and 69.

A preleukemic state in mice inoculated with Moloney murine leukemia virus (Mo-MuLV) was characterized. Six to 10 weeks after neonatal inoculation, animals developed mild splenomegaly and generalized hematopoietic hyperplasia. The hyperplasia was evident from myeloid and erythroid progenitor assays. A nonleukemogenic variant, Mo+PyF101 Mo-MuLV, did not induce the hyperplasia; this suggests that the hyperplasia is a necessary event in Mo-MuLV leukemogenesis. Another variant, MF-MuLV, which contains the long terminal repeat of Friend MuLV and causes erythroid leukemia instead of T-cell lymphoma, also induced the preleukemic hyperplasia. A model for Mo-MuLV leukemogenesis is presented in which two infection events are necessary: the first leads to generalized hematopoietic hyperplasia, and the second results in site-specific insertion and long terminal repeat activation of cellular protooncogenes.

The structural arrangement of the iron-sulfur centers in the non-heme iron-sulfur proteins has been the subject of extensive physical investigations.1-4 Particular emphasis has been placed on the "ferredoxins" which have seven or eight iron atoms and eight or more sulfur atoms per protein unit. Several workers5-7 have proposed that the iron-sulfur cluster compounds of composition [Fe4S4L4]n-, where L=S2C2(CF3)2, may be useful model systems for the ferrodoxins. We report here a brief description of the structural parameters for the Fe4S4L4-2 cluster and the Mössbauer data for the three derivatives, n=0, 1, 2. The data are compared with that reported for inorganic cluster systems of known structure and with the available data on non-heme iron-sulfur proteins.

Davis, B. R. (Communicable Disease Center, Atlanta, Ga.), and W. H. Ewing. Lipolytic, pectolytic, and alginolytic activities of Enterobacteriaceae. J. Bacteriol. 88:16–19. 1964.—Of 2,262 cultures of Enterobacteriaceae tested, only cultures of Enterobacter liquefaciens(Aerobacter liquefaciens), Serratia, Proteus vulgaris, and P. mirabilisgave evidence of lipolytic activities. None of the cultures liquefied pectate or alginate. The value of tests for these activities in diagnostic work with Enterobacteriaceae, and in defining the limits of that family, is discussed.