Effect of dietary fibre on the mutagenicity and distribution of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ)

Female BALB/c mice were fed either a fibre-free diet or one supplemented with 30% wheat-bran for 5 weeks. The ability of these mice to convert MeIQ to a bacteria mutagen in vivo was determined using intrasanguinous host-mediated bacterial mutation assays. Less mutagenic activity was detected in the livers of mice fed the bran-supplemented diet compared with those fed the fibre-free diet. Subsequent experiments demonstrated that the effect of brain was not due to modifications in hepatic metabolism, but to changes in uptake of MeIQ from the gastrointestinal tract.     

ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm³ has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 µm; it reassociates with a low C₀t_1/2 after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm³. MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm³, slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function.     

Immunochemical evidence that the single lactate dehydrogenase of lampreys is more similar to LDHB4 than to LDHA4 of hagfish

The tetrameric lactate dehydrogenases (LDH) of vertebrates contain several different subunits that arose by gene duplication. While the A and B subunits occur in all classes of gnathostomes, the enzymes of agnathans appear to represent two stages in the evolution of vertebrate LDH. Lampreys of the family Petromyzontidae have a single enzyme classified as LDHA4, while hagfish possess both A and B subunits which form only the two homopolymers LDHA4 and LDHB4. It is generally assumed that the original vertebrate LDH was an A4 type, that duplication to give the B subunit occurred prior to the divergence of lampreys and hagfish, and that modern lampreys subsequently lost expression of the B gene. Lactate dehydrogenases were purified from representatives of all three lamprey families, and it was confirmed that members of the Mordaciidae and Geotriidae also possess single tetrameric LDH enzymes containing one subunit type. The kinetic properties of the lamprey LDH enzymes were compared with the LDH homopolymers of hagfish, skate, and sardine. These properties did not allow the lamprey enzymes to be unequivocally identified as either LDHA4 or LDHB4. Immunochemical titration using antisera against lamprey and hagfish LDH homopolymers demonstrated that the lamprey LDH enzymes showed greater immunochemical similarity to LDHB4 than to LDHA4 of hagfish. It is concluded that there is little evidence for the claim that the original vertebrate LDH was an A4 rather than B4 type.     

Genetic restrictions in the development of antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 by nude mice implanted with semiallogeneic thymus glands

Athymic nude mice implanted with F1 thymus glands were used to investigate genetic restrictions regulating T cell-macrophage (M phi) interactions in the development of antibody responses to GAT. Spleen cells from conventional mice developed comparable primary plaque-forming cell (PFC) responses when stimulated by syngeneic and allogeneic GAT-M phi. However, spleen cells from strain A nude mice implanted with (A X B)F1 thymus glands were tolerant of strain B alloantigens and developed GAT-specific PFC responses to strain A GAT-M phi and allogeneic strain C GAT-M phi, but failed to respond to strain B GAT-M phi. The lack of primary GAT-specific PFC responses by spleen cells from (A X B)thy----A nude mice stimulated by strain B GAT-M phi was not due to detectable suppressor mechanisms. However, an allogeneic effect stimulated by H-2- or non-H-2-disparate GAT-pulsed or unpulsed M phi was able to overcome the inability of spleen cells from (A X B)F1 thy----A nude mice to respond to strain B GAT-M phi. Furthermore, the inability to respond to strain B GAT-M phi was overcome by the addition of supernatant fluids from independent cultures of H-2-disparate cells. These results 1) demonstrate that T cells from A nude mice implanted with (A X B)F1 thymus glands did not recognize nominal antigen in the context of B MHC antigens, and 2) suggested that the T cell repertoire was altered in strain A nude mice implanted with (A X B)F1 thymus glands, such that T cells that could recognize GAT in association with strain B MHC antigens were functionally deleted.     

Structural changes in synthetic myosin minifilaments and their dissociation by adenosine triphosphate and pyrophosphate

Morphologically similar short myosin and rod filaments (minifilaments) have been prepared in 10 mM Tris--citrate buffer, pH 8.0, in the absence of other myosin or rod forms. Both minifilament systems are dissociated in the same manner in the presence of ATP or pyrophosphate. Identical binding of these ligands to myosin and rod minifilaments suggests that myosin heads play no role in substrate-induced destabilization of the minifilaments. The effects of ATP and pyrophosphate on minifilaments are similar to their dissociating effect on synthetic filaments [Harrington, W. F., & Himmelfarb, S. (1972) Biochemistry 11, 2945--2952], thus justifying their use in conformational studies in lieu of filaments. In view of their small size and homogeneity, the minifilaments constitute an appropriate material for such studies. The binding of pyrophosphate to myosin and rod minifilaments decreases their alpha-helical content, as measured by circular dichroism. No change in the secondary structure of subfragment 1 and light meromyosin is observed upon binding of pyrophosphate, but substantial changes (10%) are detected in subfragment 2. The structural changes in myosin, possibly relevant to contraction, are localized in the subfragment 2 region of the molecule. These results emphasize the importance of charge interactions in the functional behavior of thick filaments.     

Effects of moderate short-term potassium depletion in normal humans the role of prostaglandins

The role of prostaglandins (PG) in the effects of potassium (K⁺)depletion was studied in six normal women. A mean K⁺-deficit of 220 mEq was induced with and without concomitant treatment with indomethacin (150 mg/day). Mean serum K⁺ concentration decreased from 4.2 ± (S.E.) 0.1 to 3.2 ± 0.1 mEq/L without indomethacin and from 4.1 ± 0.1 to 3.2 ± 0.1 mEq/L with indomethacin. “Supine” and “upright” plasma renin activity (PRA) and plasma norepinephrine concentration (NE) were unaltered by K⁺ -depletion alone but decreased with indomethacin. Plasma aldosterone (PA) was suppressed during K⁺-depletion (control: 7.2 ± 2.6 ng/dl supine, 19.3 ± 8.1 ng/dl upright; K⁺-depletion: 2.6 ± 0.3 ng/dl supine, 5.5 ± 1.3 ng/dl upright) and was paralleled by a decrease in urinary aldosterone. K⁺-depletion decreased urinary PGE₂ from 667 ± 133 to 343 ± 60 ng/day (P < 0.025) without a change in PGF₂. The dose of exogenous angiotensin II (A II) which increased diastolic blood pressure by 20 mm Hg (pressor dose) was 7.1 ± 1.4 ng/kg/min during control and increased to 11.0 ± 0.7 ng/kg/min during K⁺-depletion (P < 0.05). Indomethacin increased the sensitivity to A II both during control (pressor dose: 4.9 ± 0.6 ng/kg/min) and K⁺- depletion (pressor dose: 6.0 ± 1.0 ng/kg/min). These results indicate that in healthy subjects, moderate short-term K⁺-depletion does not affect PRA or NE but decreases production of aldosterone and PGE₂ by the kidney. The changes in vascular sensitivity to exogenous A II during K⁺-depletion and indomethacin and the decreases in plasma NE and PRA during indomethacin may be explained by changes in vascular vasodilator PG.     

Ribosomal proteins S3, S6, S8 and S10 of Escherichia coli localized on the external surface of the small subunit by immune electron microscopy

The three-dimensional locations of Escherichia coli ribosomal proteins S3, 86, S8 and S10 on the surface of the small subunit were determined by immune electron microscopy.All four proteins are located on the “external surface” of the small subunit; i.e. on the side of the subunit in contact with the cytosol in the 70 S ribosome. Proteins S3, S6, S8 and S10 map at single sites, although the S3 site is extended approximately 40Å along the long axis of the subunit. S8 is located near the base of the cleft separating the platform from the upper one-third or head; protein S10 is located in the head, near the site previously mapped for S14; S3 extends from the level of the constriction to near the top of the head in the vicinity of S10; and S6 is located on the platform of the small subunit near the site previously mapped for S11.The locations of these proteins correlate well with other information on their spatial relationships obtained from assembly interactions, neutron diffraction, crosslinking and protein associations.     

In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein.

Six different anti-HIV envelope antibodies and one irrelevant control antibody were coupled to ricin A chain and tested for their efficacy in inhibiting HIV tissue culture infections. The anti-HIV antibodies consisted of five monoclonals, three of murine and two of human origin, and one polyclonal preparation prepared by affinity purifying pooled serum antibodies from HIV-infected humans on rgp160. The binding specificity of the antibodies was defined by ELISA by using recombinant envelope proteins and synthetic peptides, and by flow cytometry on HIV-infected cells. The in vitro efficacy of the antibodies was tested by the abilities of the immunotoxins to inhibit protein synthesis in persistently infected cell lines and by their abilities to inhibit HIV production during both acute and persistent infection as measured with an HIV-specific focal immunoassay. The immunotoxins were tested against a panel of distinctly different HIV isolates. The results indicate the following: 1) A mAb to the immunodominant neutralizing loop was highly effective against homologous strains of HIV, but had no activity against heterologous HIV. 2) The efficacy of anti-gp41 mAb varied depending upon the epitope recognized and possibly the affinity of binding to gp41. 3) The polyclonal human anti-gp160 antibodies produced the immunotoxin with the broadest specificity for different HIV strains and the greatest specific activity. This is related to the polyclonal nature of the preparation rather than an increase in relative avidity of the antibody. 4) Activity of an immunotoxin is not a direct function of the binding of the antibody to the surface of infected cells. 5) The ability of an immunotoxin to halt the spread of infection through a tissue culture cell population is dependent upon the ability of the antibody to neutralize the virus as well as the activity of the toxin. Our data suggest that efficacious immunotoxins for the treatment of AIDS may be made with polyclonal anti-envelope antibodies derived from the serum of patients who have been infected with HIV or with appropriately chosen anti-gp41 antibodies.     

Determinants of the microbiological characteristics of South Australian swimming pools

A survey of 100 swimming pools has been conducted to assess the effectiveness of disinfection practices against various microorganisms and to check compliance with recommended chlorine levels and pH. Microbiological quality was assessed from densities of total coliforms, Escherichia coli, and Pseudomonas aeruginosa, total colony counts, and the presence or absence of amoebae, including the pathogen Naegleria fowleri. Although a free chlorine residual of 1.0 mg/liter and a pH range of 7.0 to 7.6 are recommended by local health authorities, 41 pools had a lower free chlorine residual and 37 had a pH outside this range. Multiple logistic regression analysis was used to test the association of field measurements with the microbiological data. The analysis demonstrated a strong positive association of free chlorine with bacteriological quality and the absence of Naegleria spp. No other field measurement was predictive in this regard, although the absence of all amoebae was associated with a relatively low water temperature and pH. Using a mathematical model derived from this analysis, it was estimated that 99% of pools would have acceptable bacteriological quality and 94% would be free of Naegleria spp. at a free chlorine residual of 1.0 mg/liter. However, at the mean water temperature (23 degrees C) and pH (7.5) seen in this study, other amoebae would still be detectable in 500-ml samples taken from 40% of pools at this chlorine level.     

Effect of antihypertensive therapy on sympathetic nervous system activity in patients with essential hypertension

The sympathetic nervous system (SNS) plays a major role in blood pressure regulation. Although the exact relationship of the SNS to the etiology of hypertension remains undetermined, many of the agents used to treat hypertension interfere with this system. Clonidine, methyldopa, guanethidine, and reserpine decrease SNS tone whereas hydralazine, minoxidil, and hydrochlorothiazide increase it. Most evidence suggests that beta-adrenergic blocking agents decrease SNS activity. The effect of prazosin and captopril on the SNS requires further study. The appropriate use of these antihypertensive agents requires a knowledge of their sites of action and the physiological reflexes they induce. Efficacy, toxicity, and effective drug combinations can be predicted based on their mechanism of action and effect on SNS activity.