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Oxytetracycline-resistant (OTcr) and tetracycline-resistant (Tcr) Aeromonas hydrophila were isolated commonly from catfish intestinal contents and the water and sediment of catfish culture ponds, but less frequently in market catfish. Isolates demonstrated two resistance phenotypes, Tcr OTcr and Tcs OTcr, when plated directly on to MacConkey agar containing 30 micrograms of tetracycline or oxytetracycline per ml. Tcs OTcr isolates expressed Tcr after induction by 1 h of growth in tryptic soy broth containing 1 microgram of tetracycline per ml. Over 90% of the resistant aeromonads hybridized with DNA probes for class A or class E Tcr determinants; class E was twice as prevalent as class A. The distribution of class A and E Tcr determinants varied with the Tcr phenotypes. Prior to induction, 86% of isolates with class A determinants were Tcr as well as OTcr, while only 16% with class E determinant were Tcr. Three of 5 Tcr aeromonads without class A or class E determinants and 1 of 14 with class E determinants transferred Tcr to Escherichia coli.

Escherichia coli containing the cryptic tetracycline resistance determinant (class F) from the Bacteroides fragilis transposon Tn4400 on plasmid pGAT400 expressed a detoxification of tetracycline as well as an active efflux of tetracycline. This finding concurs with the report of detoxification for a related tetracycline resistance determinant from B. fragilis on Tn4351 (B. S. Speer and A. Salyers, J. Bacteriol. 170:1423-1429, 1987), which specifies a 10-fold-higher resistance than Tn4400. Inactivation of tetracycline occurred at an initial rate of congruent to 0.7 micrograms of tetracycline per h per 10(8) cells, as determined by biologic assay and chromatographic analysis. The detoxification is a chemical degradation which can occur in the absence of energy-dependent efflux. The products of this degradation were not substrates for active transport into susceptible cells or out of pGAT400-containing E. coli. These results indicate that Tn4400 mediates two functionally different mechanisms for tetracycline resistance: an active efflux of tetracycline and a degradation of tetracycline.

The structural and regulatory tetracycline resistance genes of transposon Tn10 are located on a 2,700-base pair HpaI fragment. We have used eight tetracycline-sensitive mutations in the 2,700-base pair fragment, cloned into two compatible plasmids, to demonstrate that two complementation groups are required for tetracycline resistance. By genetic recombination with plasmids containing the regulatory or structural regions for resistance, we have determined that both complementation groups reside within the structural region. The complementation groups, designated tetA and tetB, are proximal and distal, respectively, to the promoter for the tetracycline resistance structural region. The tetB mutations are in the portion of the structural region that is known to encode the 36,000-molecular-weight, inner-membrane TET protein. The levels of tetracycline resistance expressed during complementation suggest a complex interaction between the products of the tetA and tetB loci.

It has been proposed that measurements of plasma dopamine-beta-hydroxylase (DBH) may allow for assessmetn of adrenergic tone and may elucidate a possible neurogenic contribution to essential hypertension. We performed a series of measurements of DBH in fifty-seven normotensive and fifty hypertensive black and white men in order (1) to compare DBH to selected blood pressure patterns and (2) to evalutate the influence of salt intake, posture and race on plasma DBH. Plasma DBH, measured on unrestricted salt intake with subjects supine, was 42 +/- 4 Units/L in white normotensives, greater (P less than 0.05) than black normotensives (26 +/- 6 Units/L). White hypertensives had greater plasma concentrations of DBH than black (35 +/- 3 VS. 24 +/- 5, P less than 0.05). Normotensives did not differ from hypertensives. Dietary salt restriction and upright ambulation increased plasma DBH activity in hypertensives. Although DBH did not correlate directly with blood pressure, high DBH values were associated with lability of blood pressure in hypertensives but not in normotensives. There are two possible explanations for our results: (1) multiple factors influence plasma DBH activity and plasma levels reflect more than adrenergic function, or (2) essential hypertension is a multifactorial disease and excess sympathetic neuronal activity alone is not sufficient to produce sustained hypertension.

We studied the plasmid and antibiotic resistance characteristics of 35 strains of Enterobacteriaceae recovered from faecal specimens of children with diarrhoea in Central General Hospital, Bandung, Indonesia. Twenty three Escherichia coli, three Providencia, three Proteus, three Klebsiella, two Enterobacter and one Citrobacter were examined. All strains were multiply resistant, many carrying six to nine antibiotic resistances. Most of these resistances were transferable to a laboratory E. coli strain and were carried on large-sized plasmids. All recently-described tetracycline resistance determinants (Classes A----D) were represented; the most common was the Class B, or TN10 type. The TEM-1 beta-lactamase was detected in 17 out of 21 ampicillin-resistant strains examined. The OXA-1, PSE-1, and SHV-1 enzymes were also found. Of 23 plasmids tested, all could be classified into one of eight different incompatibility groups: IncFII, IncN, IncB, IncF1, IncI1, IncI2, IncH2 and IncT. These studies demonstrate the existence of large multiresistant transferable plasmids representing common incompatibility groups and bearing common tetracycline and ampicillin resistance determinants in enteric strains isolated from children hospitalized in Indonesia.

Survival in the mouse and human intestine of Escherichia coli host-vector systems used and proposed for recombinant DNA technology was assessed. There was no detectable survival of severely disabled E. coli K12 strain X1776 in mice or in human subjects 24 hours after ingestion. The same strain bearing the plasmid pBR322, however, was recovered from human subjects for 4 days in amounts of six organisms for every million ingested. Nondisabled E. coli K12 strain X1666, with or without pBR322, survived in 10(4)-fold greater numbers and for 2 days longer, with better recovery of the plasmid-containing derivative. Although the plasmid-bearing strains were recovered for longer periods, no intestinal colonization was noted. Despite the presence of pBR322 for a maximum of 6 days in the human intestine, there was no evidence that it was transferred from either bacterial host to endogenous aerobic fecal bacteria.

The inner membrane TET (TetA) protein, which is involved in Tn10-mediated microbial tetracycline resistance, consists of two domains, alpha and beta, both of which are needed for tetracycline resistance and efflux (M.S. Curiale, L.M. McMurry, and S.B. Levy, J. Bacteriol. 157:211-217, 1984). Since tetracycline-sensitive mutants in one domain can partially complement sensitive mutants in the other domain and since some sensitive mutants show dominance over the wild type, a multimeric structure for TET in the membrane had been suggested. We have studied this possibility by using tetA-phoA gene fusions. We fused all but the last 40 base pairs of the tetA gene with the carboxy terminus of the phoA gene for alkaline phosphatase (PhoA), whose activity requires its dimerization in the periplasm. The tetA-phoA fusion protein was under control of the tetracycline-inducible regulatory system for the tetA gene. Induction led to the synthesis of a 78,000-dalton inner membrane protein. Tetracycline resistance was expressed at reduced levels, consistent with the terminal beta domain deletion. Alkaline phosphatase activity was also present, but at low levels, suggesting that some, but not all, of the fusion proteins had their carboxy-terminal ends in the periplasm. When wild-type or mutant TET proteins were present in the same cell with the fusion protein, the tetracycline resistance level was affected (raised or lowered); however, phosphatase activity was reduced only when TET proteins with intact or near-intact beta domains were present. These findings suggest that TET functions as a multimer and that intact beta domains, on TET molecules in the heterologous multimer, either allow fewer PhoA moieties to project into the periplasm or sterically hinder PhoA moieties from dimerizing.

The plasmid pUC18 contains a lacZ alpha-complementation gene that codes for a small peptide that can complement the delta M15 mutation of the Escherichia coli lacZ (beta-galactosidase) gene, converting bacteria carrying that mutated gene from the lacZ- to the lacZ+ phenotype. This plasmid was used in experiments designed to study mutagenesis by human neutrophils. E coli carrying pUC18 were incubated with neutrophils under conditions in which little ingestion of the bacteria took place; the plasmid was then isolated and transformed into an E coli strain (BOZO) that carries the lacZ delta M15 mutation. Of these transformants, 11 of 205,000 were lacZ, suggesting that in these 11, alpha-complementation had been lost through a mutation. No lac- colonies were detected among several hundred thousand BOZO transformed with plasmid isolated from incubations in which phagocytosis could take place, nor from incubations from which neutrophils were omitted. Despite the lac- phenotype of these 11 transformants, plasmids reisolated from nine of them showed normal alpha-complementing ability when transformed into fresh BOZO. These findings indicated that in these nine, the mutations were located in the chromosomes of the transformed BOZO. It thus appears that on exposure to activated neutrophils, a plasmid may acquire a lesion (? mutation) that can somehow be transferred to the genome of a recipient microorganism, resulting in repair of the damaged plasmid accompanied by mutation of the recipient's chromosome. Restriction mapping of the DNA from four of these nine chromosomal mutants suggested that the mutations did not represent major insertions or deletions in the portion of the bacterial chromosome corresponding to the pUC18 lac operon insert, nor in the remainder of the lacZ delta M15 gene. These results confirm previous work showing that exposure to activated neutrophils can induce mutations in biological systems, and provides an experimental model in which the mechanism of neutrophil-mediated mutagenesis may be examined.

Plasmids which specify resistance to tetracycline offer much less resistance to its more lipophilic analog, minocycline. Resistance to minocycline varies for different plasmids. In the case of plasmid R222 (bearing the class B tetracycline resistance determinant on Tn10), minocycline resistance is comparatively high (10 microgram/ml, or 6% of the tetracycline resistance level). For plasmid pIP7 (bearing the class A determinant), minocycline resistance is only 1% of the tetracycline resistance level. To understand the basis for these differences, we compared the transport of the two tetracyclines by susceptible cells and by resistant cells. Uptake of minocycline by susceptible cells was 10 to 20 times more rapid than uptake of tetracycline and occurred largely via an energy-dependent route. This host-mediated energy-dependent uptake of both analogs was still present in tetracycline-resistant cells. In resistant cells, the same plasmid-mediated active efflux system previously described for tetracycline also exported minocycline. The 15-fold greater susceptibility of tetracycline-resistant R222-bearing cells to minocycline as compared with tetracycline could be explained at least in part by the more rapid influx of minocycline, which more easily overcame the efflux system. The particularly low minocycline resistance offered by pIP7 was due to a weak efflux for minocycline, 10-fold less effective than that mediated by R222. The rate-limiting step for uptake of both analogs appeared to be the outer membrane. That the lipophilic minocycline should cross this membrane more rapidly than tetracycline stands in contrast with other studies which show the outer membrane to be a barrier for entry of lipophilic substances.

Changes in hemodynamic variables and plasma renin activity (PRA) induced by short-term alteration of blood pressure and sympathetic nervous system activity were studied in normotensive and hypertensive men on high-sodium and low-sodium intake. Blood pressure and heart rate alterations caused by standard cold pressor tests, amyl nitrite inhalation, phentolamine infusion, and phenylephrine infusion were recorded. PRA was measured at time 0, 5, 15, and 30 min. No change in PRA occurred in either group following cold pressore test and amyl nitrite inhalation. Phenylephrine suppressed PRA in normal but not hypertensive subjects, and phentolamine elevated PRA in normotensive and hypertensive subjects regardless of diet. Short-term stimulation of the sympathetic nervous system does not appear to elevate PRA in man. Suppression of PRA by an alpha agonist was not observed in hypertensives and may reflect barorecptor insensitivity in this group of subjects.

A fifth tetracycline(Tc)-resistance determinant, designated class E, has been identified on a transferable plasmid found in a fecal strain of Escherichia coli. This determinant does not show homology by DNA-DNA hybridization at high stringency with any of four other Tc resistance determinants (classes A, B, C and D) previously described among the Enterobacteriaceae. Resistance is inducible by 1 microgram Tc/ml and increases the minimum inhibitory concentration 130-fold for Tc and 3.5-fold for minocycline. The mechanism, like that of the other four determinants examined, appears to involve an active efflux of the drug. Using a 32P-labeled cloned fragment containing the resistance determinant, we have found the determinant in Aeromonas, but not in any of over 200 other E. coli strains tested.

A Ca2+, Mg2+-dependent endonuclease activity was detected in erythroleukemic cells undergoing differentiation in vitro in response to induction by dimethyl sulfoxide (Me2SO) or hexamethylene-bis-acetamide (HMBA). The endonuclease activity was demonstrated in isolated nuclei within 6 hr after the addition of inducer, reached maximum levels between 24 and 48 hr, and returned to control levels within 72 hr. The activity caused single strand breaks in high molecular weight native DNA, which could be labeled at exposed 3'-OH termini with Escherichia coli DNA polymerase I and radiolabeled nucleotides. Alkaline elution studies revealed DNA fragmentation that appeared coincident with the presence of the endonuclease activity. The detection and levels of single strand DNA breakage correlated with induction of terminal differentiation by Me2SO or HMBA. Induction of the endonuclease activity was reversible: depletion of Me2SO from the growth medium after treatment for 6 and 18 hr led to a rapid decrease in the level of activity. Removal of the inducer prevented terminal differentiation, a finding that strongly suggests the endonuclease activity is present during the precommitment phase of differentiation. DNA fragmentation was not observed in cells incubated with hemin, which has been shown previously to increase the cytoplasmic level of globin mRNA without causing commitment to terminal maturation. Me2SO did not induce the endonuclease activity or DNA fragmentation in an uninducible Friend cell line.

A histological stain described by Menzies in 1963 has been useful in detecting early evidence of erythroleukemia (Friend disease) in mouse hemopoietic tissues fixed in formaldehyde solution. With the use of this stain, one can distinguish erythroblastic leukemia cells from lymphoblastic leukemia cells.

Friend virus infection of susceptible mice led rapidly to fulminant erythroleukemia and death. Subcutaneous implantation of leukemia spleen bits into splenectomized normal animals led to their early death from Friend leukemia. In contrast, bits of leukemic spleen implanted sc into splenectomized leukemic mice prolonged the survival of these animals. Concomitant with this survival was a reversal of the virus-induced immunosuppression and an increase in the levels of circulating, neutralizing, antivirus activity. This marked difference in response to leukemic spleen implants by leukemic as compared to normal mice reflected previous contact of the former with Friend Virus. Our studies indicated that the Friend virus-infected mouse mounted a resistance to the virus infection, which under certain conditions is capable of reversing the disease process.

To learn more about the regulation of blood pressure in renal parenchymal disease, 57 subjects (18 normal controls, 25 patients with essential hypertension and 14 with renal parenchymal disease and hypertension) were evaluated for peripheral renin activity, 24-hour urinary kallikrein activity and whole-blood volume. Blood volumes were significantly lower in patients with essential hypertension (P less than 0.001) and those with renal disease and hypertension (P less than 0.001) than in normotensive subjects. Renin activities (measured after the subjects were standing) were also lower in patients with essential hypertension and hypertension due to renal disease (P less than 0.01 and P less than 0.02, respectively). Kallikrein activity was similar in subjects with renal disease and those with hypertension (P less than 0.05) but markedly diminished in both groups as compared with normotensive subjects (P less than 0.001 and P less than 0.01, respectively) when glomerular filtration rates were taken into account. The kallikrein-kinin system may be involved in the hypertension associated with renal parenchymal disease.

MRB is a major R222 plasmid-encoded outer membrane protein previously described by this laboratory which is immunologically identical with F plasmid traTp. We have detected an apparent precursor polypeptide of MRB in Escherichia coli minicells which reacts with specific anti-MRB serum. In the presence of proteolytic inhibitors, immunologically reactive precursor MRB polypeptides are found in whole cells and minicells containing R222 or F plasmids. When R222-containing minicells were incubated for short periods of time with [35S]methionine, the precursor was predominantly labeled but was shown to be converted into MRB during subsequent incubation in media containing nonradioactive methionine. This conversion was inhibited in the presence of the proteolytic inhibitor tosyllysylalanyl chloramethyl ketone. These studies show that at least one plasmid-encoded membrane protein is processed in its E. coli host in a way resembling that of host cell membrane proteins.

A major portion of tetracycline accumulation by susceptible bacterial cells is energy dependent. Inner membrane vesicles prepared from susceptible Escherichia coli cells concentrated tetracycline 2.5 to 5 times above the external concentration when the electron transport substrate D-lactate or reduced phenazine methosulfate was added. This stimulation was reversed by cyanide, 2,4-dinitrophenol, and carbonyl cyanide m-chlorophenyl hydrazone. These vesicles data showed that proton motive force alone could energize tetracycline uptake. The lactate-dependent uptake had a pH optimum of 6.9 and a magnesium optimum of 1 mM and was not saturable up to 400 microM tetracycline. Although the vesicles were not as active as cells in concentrating tetracycline, they were less active to a similar extent in concentrating tetracycline, they were less active to a similar extent in concentrating proline, the transport of which is known to be solely proton motive force dependent. Therefore, we concluded that the active uptake of tetracycline in susceptible cells was largely, if not solely, energized by proton motive force.

Pieces of Friend leukemic spleens implanted sc into mice regenerated into normal-appearing spleens if animals were protected against the leukemia. In unimmunized animals, the implant regenerated normally, but was subsequently infiltrated by leukemia cells. This leukemia cell infiltration required at least 30% stromal regeneration of the implant. The hematopoietic regeneration was primarily a repopulation with cells from the host animal, not with cells indigenous to the donor spleen. The development of the implant, whether normal or leukemic, was retarded by the presence of the host spleen, whether leukemic or normal. These studies strongly suggested that the stromal cells of the spleen are not directly involved in the virus-induced leukemic process but acted as supporting structure for the malignant cells.

Human hypertension has been related to abnormalities of autonomic blood pressure regulation. In order to characterize a possible defect, we have studied several aspects of reflex autonomic circulatory control in normal subjects, patients with primary hypertension, and two types of subjects with uremia and elevated blood pressure. Phenylephrine infusion (a measurement of baroreceptor response to high pressure stimulus) resulted in similar hemodynamic changes in all types of subjects. However, all groups of patients exhibited significantly different hemodynamic responses to amyl nitrite inhalation (an index of baroreceptor response to low pressure stimulus). These results suggest that patients with uremia and those with primary hypertension differ from each other and normal subjects regarding baroreceptor reflex function. Furthermore, one type of uremic patient may have a neurogenic component to their hypertension similar to experimental animals following surgical section of afferent baroreceptor nerves.

We have used antibodies directed against two histone H2a variants, H2a.1 and H2a.2, to probe chromatin structure in Friend erythroleukemia cells. Each molecule has at least one unique antigenic determinant, as well as determinants shared by both variants. Some Friend leukemia cell types contained H2a molecules which showed altered immunologic reactivity with the two antisera. The accessibility of the H2a variants in chromatin to anti-H2a antibody was different as measured by the use of whole chromatin as an immunoabsorbent and by binding of antibody to nucleosomes in a solid phase radioimmunoassay. While anti-H2a.1 IgG bound to chromatin, anti-H2a.2 IgG did not. Moreover, anti-H2a.1 IgG binding to chromatin from different Friend cell types reflected, in general, the relative amounts of H2a.1 in total chromatin. The different reactivity of the two antisera with chromatin was also observed with isolated nucleosomes: anti-H2a.1 IgG bound but anti-H2a.2 did not. Furthermore, the binding of anti-H2a.1 Ig with subfractions of nucleosomes varied; H1-depleted, high mobility group-enriched nucleosomes reacted better than H1-containing, high mobility group-depleted nucleosomes. These findings demonstrated a heterogeneity in the organization of H2a variants in chromatin within nucleosomal subfractions of chromatin and among chromatin of different Friend leukemia cell types. Moreover, most of the antigenic determinants common to both H2a variants were shown to be buried within the nucleosome core; only H2a.1-unique determinants were accessible to an anti-H2a.1 IgG molecule.

The survival of a laboratory strain and a naturally occurring fecal strain of Escherichia coli, with and without a Tn5-containing derivative of ColE1, was examined after aerosol dispersal in a laboratory office and a barn under ambient temperature and humidity conditions. Following the release of paired strains, air and diverse types of surfaces were assayed for the test organisms. In both environments, the number of airborne bacteria declined rapidly within the first 2 h. Longer survival was found on surfaces and varied with surface type: recovery was greatest from wood products. Organisms persisted for 1 day in the office and for up to 20 days in the barn. Survival of the fecal strain was better than that of the laboratory strain in both test environments. In general, plasmid-bearing strains fared similarly to their plasmidless parents, but in several comparisons the ColE1::Tn5-containing strain showed enhanced survival. These studies have implications for the present and proposed release of genetically engineered organisms with and without plasmid vectors.

Levy, Stuart B. (Institut du Radium, Paris, France). Isologous interference with ultraviolet and X-ray irradiated bacteriophage T(2). J. Bacteriol. 87:1330-1338. 1964.-Qualitative and quantitative analysis of the interference capacity of an irradiated T(2) bacteriophage was made with ultraviolet and X-ray irradiation. Two different effects were found to explain the total interference picture in the ultraviolet-irradiated system: exclusion and depression. Exclusion is the absolute inhibition of infectious phage growth in the bacterial host. Depression is the diminution of burst size in instances where the infectious phage has not been excluded. Both effects were seen when the infectious phage was added after the addition of ultraviolet-irradiated phage. Doses between 1,600 and 2,200 ergs/mm(2) (survivals, ca. 10(-7)) showed the greatest exclusion effect (70%). Exclusion was lost between 6,500 and 7,500 ergs/mm(2). The depression effect was highest (90%) at lower doses (survivals, ca. 10(-6)), falling off as the dose range went above 1,600 ergs/mm(2) or survivals of 10(-7). Depression was lost at 3,000 ergs/mm(2). X-ray irradiation (both direct and indirect) to survivals less than 10(-2) showed no interference capacity in the phage irradiated. Indirect X-ray irradiation to survivals between 5 and 10% showed 50% exclusion, but no depression.

Penicillin has been used to purify large quantities of minicells.

Salmonella enteritidis(14)C-endotoxin was recovered predominantly from the nuclear and mitochondrial subcellular fractions of livers and spleens of mice and rats, 3.5 hr and 3 days after intravenous administration. Of the recovered radioactivity, 10 to 20% was present in the liver mitochondrial fraction as high-molecular-weight, biologically active material, suggesting the presence of intact endotoxin. Autoradiographic studies demonstrated nuclear and cytoplasmic labeling in the liver and at least nuclear label in spleen cells. The resistance of rats, as compared to mice, to the induction of amyloidosis does not appear to be based on a difference in subcellular localization of endotoxin within the reticuloendothelial system.

Both fi(+) and fi(-) type R factors function as intact replicative units when segregated into Escherichia coli minicells. Hybridization studies demonstrate that at least 95% of the deoxyribonucleic acid (DNA) in R(+) minicells is episomal in origin. About half of the DNA can be extracted in a closed circular form and about 75% of the DNA is membrane associated. DNA, ribonucleic acid, and protein synthesis proceeds in R(+) minicells in contrast to R(-) minicell controls. The system offers a unique opportunity to study a relatively small replicative unit in a native cell environment and a simple means of isolating large quantities of episomal DNA.