DNA looping by the Sfi I restriction endonuclease

The SfiI endonuclease has to interact with two copies of its recognition sequence before it can cleave DNA. To demonstrate that the reaction of SfiI on a DNA with two sites involves the formation of a DNA loop, and to characterise the looping interactions on supercoiled and linear DNA, a series of plasmids was constructed with lengths of DNA between two SfiI sites varying from 104 to 211 bp. Both supercoiled and linear forms of each DNA were tested as substrates for SfiI. The reactions were monitored from the rates of DNA cleavage and from the generation of partially cleaved products, the latter indicating loop disruption before cleavage of both sites. On both supercoiled and linear DNA, the stabilities of the complexes spanning two SfiI sites varied in sinusoidal fashion with the distance between the sites, in the manner characteristic of a process governed by the helical periodicity of DNA. In all cases, the looping interaction was stabilised by DNA supercoiling. The sinusoidal variation from SfiI reactions on supercoiled DNA at 50 degreesC yielded a helical repeat of about 11.5 base-pairs per turn.     

Domain-swapping analysis of FtsI, FtsL, and FtsQ, bitopic membrane proteins essential for cell division in Escherichia coli

FtsI, FtsL, and FtsQ are three membrane proteins required for assembly of the division septum in the bacterium Escherichia coli. Cells lacking any of these three proteins form long, aseptate filaments that eventually lyse. FtsI, FtsL, and FtsQ are not homologous but have similar overall structures: a small cytoplasmic domain, a single membrane-spanning segment (MSS), and a large periplasmic domain that probably encodes the primary functional activities of these proteins. The periplasmic domain of FtsI catalyzes transpeptidation and is involved in the synthesis of septal peptidoglycan. The precise functions of FtsL and FtsQ are not known. To ask whether the cytoplasmic domain and MSS of each protein serve only as a membrane anchor or have instead a more sophisticated function, we have used molecular genetic techniques to swap these domains among the three Fts proteins and one membrane protein not involved in cell division, MalF. In the cases of FtsI and FtsL, replacement of the cytoplasmic domain and/or MSS resulted in the loss of the ability to support cell division. For FtsQ, MSS swaps supported cell division but cytoplasmic domain swaps did not. We discuss several potential interpretations of these results, including that the essential domains of FtsI, FtsL, and FtsQ have a role in regulating the localization and/or activity of these proteins to ensure that septum formation occurs at the right place in the cell and at the right time during the division cycle.     

The nutrition implications of cardiac cachexia

Weight loss is a frequent sequela in patients with congestive heart failure and is commonly referred to as cardiac cachexia. This weight loss is unlike that seen in simple starvation because it preferentially involves the depletion of lean body mass. In addition, the presence of cardiac cachexia can have profound clinical implications for patients in terms of complications, clinical outcome, and overall cost. The mechanism for the alterations in body composition is multifactorial, but a major cause may be the cytokine-mediated host response to the underlying disease. This article reviews the syndrome of cardiac cachexia in light of recent evidence regarding the role of cytokines, as well as potential therapies.     

Mechanism of suppression of cell-mediated immunity by measles virus

The mechanisms underlying the profound suppression of cell-mediated immunity (CMI) accompanying measles are unclear. Interleukin-12 (IL-12), derived principally from monocytes and macrophages, is critical for the generation of CMI. Measles virus (MV) infection of primary human monocytes specifically down-regulated IL-12 production. Cross-linking of CD46, a complement regulatory protein that is the cellular receptor for MV, with antibody or with the complement activation product C3b similarly inhibited monocyte IL-12 production, providing a plausible mechanism for MV-induced immunosuppression. CD46 provides a regulatory link between the complement system and cellular immune responses.     

Serum HIV-1 RNA levels and time to development of AIDS in the Multicenter Hemophilia Cohort Study

Normal women produce small amounts of active androgens. When androgen levels are elevated, such as for example in the polycystic ovary syndrome, this is followed by the development of male physical characteristics and muscle mass, structure and function as well as android adipose tissue distribution and function. Psychological features and stress reactions also seem similar to those of men. Such women have an increased risk of developing hypertension, non-insulin-dependent diabetes mellitus and cardiovascular disease. Recent data have shown that these physical, and psychological characteristics, as well as risk of ill health, are also found in the population of women selected at random. Women in the lowest quintiles of levels of sex-hormone-binding globulin--an indicator inversely related to active androgens--are at risk of developing hypertension, non-insulin-dependent diabetes mellitus and cardiovascular mortality. The mechanism probably includes muscular insulin resistance, following a relative androgen excess. It is thus apparent that androgens, even within the highest levels of the nonselected population of women, are powerful predictors of serious disease development. The population at risk might be as large as about 20% of middle-aged women. This is an area of female disease risk which requires more attention in screening and intervention procedures.     

DNA polymerase of Dictyostelium discoideum

Only one molecular weight species of DNA polymerase was found in different developmental stages of the eukaryotic microorganism Dictyostelium discoideum. The molecular weight of this DNA polymerase is estimated to be about 127 000 by sucrose gradient centrifugation. The enzyme is present in all stages of growth and development, including dormant spores. All DNA polymerase activity is lost upon incubation of the crude extract with N-ethylmaleimide. The reaction properties, molecular weight and N-ethylmaleimide sensitivity of the D. discoideum DNA polymerase are similar to those of the DNA polymerase-alpha from mammalian sources.     

Responses of visual, somatosensory, and auditory neurones in the golden hamster's superior colliculus

1. The response characteristics of visual, somatosensory, and auditory neurones in the golden hamster's superior colliculus were investigated.2. As has been noted for other mammalian species, a distinct difference between the functional organizations of the superficial and deeper layers of the superior colliculus was observed.3. Neurones in the superficial layers were exclusively visual, with small receptive-fields, and generally did not show response decrements with repeated stimulation. The sizes of the receptive-fields did not vary appreciably as a function of retinal eccentricity.4. In the deeper layers, visual receptive-fields were large, or could not be accurately delimited, and response habituation was often evident. In addition, many cells in the deeper layers of the colliculus responded only to somatosensory stimuli. Far fewer cells, which appeared to be confined to the caudal portions of the colliculus, responded to auditory stimuli. Polymodal cells were also encountered.5. Selectivity to opposing directions of movement was tested for ninety-four visual cells. Using a ;null' criterion, 27.7% of these cells were judged to be directionally selective. A distribution of the preferred directions of these cells showed a significant preference for movement with an upper-nasal component. With a statistical criterion, 60.6% of these cells were considered to show a significant asymmetry in responding to movement in opposing directions.6. Directional selectivity was also tested for ninety-two cells following acute, unilateral, lesions of the visual cortex. For the eighty cells recorded, homolateral to the ablated cortex, 27.5% were judged as directionally selective using the statistical criterion, while 12.5% were selective with the ;null' criterion. Of the twelve cells isolated in the colliculus, contralateral to the lesions, seven were judged as directionally selective with the statistical, and three with the ;null' criterion.7. The effects of visual cortical lesions upon directional selectivity appeared to be confined to cells in the superficial layers of the colliculus. It was suggested that directional selectivity of many cells in the superficial layers of the tectum of the hamster is organized cortically.8. A clear spatial correspondence was observed for the receptive-fields of visual, somatosensory, and auditory neurones.9. As has been suggested for other species, the hamster's superior colliculus appears to play an important role in orienting the animal toward visual, somatosensory, and auditory stimuli.     

Preferential utilization of ketone bodies for the synthesis of myelin cholesterol in vivo

1. The distribution of radioactivity among lipid classes of myelin and other subcellular brain fractions of young rats (18-21 days) was determined after in vivo injection of (3-(14)C-labelled ketone bodies, [U-(14)C] glucose or [2-(14)C] glucose. 2. The incorporation ratios (sterol/fatty acids) were 0.67, 1.48, 0.25, 0.62 and 0.54 for whole brain, myelin, mitochondria, microsomes and synaptosomes, respectively, with (3-(14)C)-labelled ketone bodies as substrate and 0.37, 0.89, 0.19, 0.34 and 0.29 with [U-(14)C] glucose as substrate. These data show that, both in whole brain and in subcellular brain fractions, acetyl groups derived from ketone bodies are used for sterol synthesis to a large extent than acetyl groups originating from glucose. 3. The specific radioactivity of cholesterol is much higher in myelin than in whole brain or in the other brain fractions, particularly after administration of labelled ketone bodies as substrate. 4. The incorporation patterns of acetoacetate and D-3-hydroxybutyrate were very similar, indicating that both ketone bodies contribute acetyl groups for lipid synthesis via the same metabolic route. 5. Our data suggest that a direct metabolic path from ketone bodies towards cholesterol exists - possibly via acetoacetyl-CoA formation in the cytosol of brain cells - and that this process is most active in oligodendrocytes.