Histone acetylation and heterochromatin content of cultured Peromyscus cells

In order to explore the relationship between unacetylated arginine-rich histones and condensed chromatin structure, the extent of histone acetylation was examined in cultured cell lines derived from three species of deer mice. These species differ considerably in their genomic content of heterochromatin but contain essentially the same euchromatin content. Cells of Peromyscus eremicus, containing 34–36% more constitutive heterochromatin than Peromyscus boylii or Peromyscus crinitus cells were found to contain 28–35% more unacetylated histone H4, 22–29% more unacetylated histone H3, and 18–22% more unacetylated histone H2B. This relationship between unacetylated histones and heterochromatin content was further explored by inducing hyperacetylation of P. eremicus and P. boylii histones through treatment of cells with 15 mM sodium butyrate for 24 h. It was found that the percentages of unacetylated histones H3 and H4 remaining after butyrate treatment were proportional to the amount of constitutive heterochromatin in the genome. These data support the concept that a small core of histones in constitutive heterochromatin is inaccessible to acetylation. It was also found that the acetylated state of isolated histones was sensitive to the method of histone extraction. Thus concern must be given to preparative procedures when studying histone acetylation in order to minimize these acetate losses.     

A surprising new protein superfamily containing ovalbumin,antithrombin-III,and alpha1-proteinase inhibitor

A distant relationship between chicken ovalbumin and two human plasma protease inhibitors was revealed by computer analyses. We propose a new protein superfamily containing at least three families: ovalbumin (and probably gene X and gene Y proteins), antithrombin-III, and alpha₁-proteinase inhibitor. Although these families may have diverged from a common ancestor more than 500 million years ago, they may still share similarity in gene structure as well as in protein sequence.     

The effects of antimicrotubule agents on cell motility in fibroblast aggregates

Spherical aggregates of chick heart, sclera and skin fibroblasts were fused with tritiated thymidine-labelled aggregates of the identical cell type. After being placed in contact, the two aggregates cohered and broadened the area of contact to form a single aggregate with a planar interface between the labelled and unlabelled halves. The motility of cells in the aggregate was determined by measuring the movement of labelled cells across the boundary into the unlabelled half. Exposure to pharmacological doses of antimicrotubule agents resulted in a significant reduction in fibroblast motility in the three-dimensional aggregates.     

Development of wheat bulb fly (Della coarctata fall.) larvae and pupae at different temperatures

When eggs of wheat bulb fly were added to young winter wheat plants in pots and kept at 5°, 8°, 10°, 12°, 15° and 20°, only 14%–44% of the eggs added produced larvae that succeeded in invading plants, but when newly hatched larvae were added directly 54%–92% were successful. At temperatures from 5° to 25°, the number of days required for larvae to complete their development in well-grown plants ranged from 11–14 days at 25° to 55–80 days at 5°. This corresponds to an accumulated temperature of approximately 286 day degrees C above the developmental threshold of 0.5°.The daily rate of development of larvae was marginally most rapid between 12° and 18°. When larvae were feeding in small, poorly growing plants, development was delayed and more shoots were needed before pupation than for healthy plants.The pupal stage which develops in the field from the end of April to June needs approximately 400 and 420 day degrees for completion in males and females (threshold 5°). Total day degrees required for the hatching of the egg to adult are about 700 (males) and 720 (females).Records of maximum and minimum temperatures from a meteorological screen 1 m above the ground can be used to calculate the number of day degrees accumulated from 1 May to 12 June, from which the dates of peak emergence of wheat bulb fly can be predicted. If 350 day degrees or more accumulate during this period, the peak emergence occurs near 20 June and with 200 or fewer day degrees it occurs near 11 July.The amount of damage to wheat by wheat bulb fly larvae in 1953, 1954, 1965 and 1966 depended largely on the number of eggs laid, the date of sowing, and also on the rate at which temperature accumulated in the autumn and winter; in all years, late sown crops would have had little opportunity to grow beyond the susceptible stage by the time they were attacked.

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Résumé Quand des jeunes plants de blé d'hiver en pots sont contaminés avec des oeufs de Delia coarctata et maintenus à des températures respectives de 5°, 8°, 10°, 12°, 15° et 20° C, seulement 14% à 44% de ces oeufs donnent des larves qui parviennent á attaquer les plants, alors que si on utilise directement des larves nouveau-nées on obtient 54 à 92% d'attaques réussies. Aux températures de 5° à 25° le temps nécessaire aux larves pour achever leur développement dans les plants bien développés avant de gagner le sol pour la pupaison, est de 11 à 14 jours à 25° contre 55 à 80 jours à 5°.Une courbe de la vitesse de développement établie à partir de la durée du stade larvaire de l'éclosion à la pupaison aux differentes températures montre que le développement est le plus rapide entre 12° et 18° et que le zéro de développement est aux alentours de 0,5°. Le nombre de degrésjours accumulés par la larve de l'éclosion à la pupaison est d'environ 286. Quand les larves se nourrissent aux dépens de plants petits à croissance faible, leur développement est retardé et exige plus de temps et plus de plantes avant la pupaison que lorsqu'elles se nourrissent sur des plants vigoureux.Le stade pupal qui intervient dans la nature de fin avril à juin, nécessite approximativement 400 à 420 degrés-jours pour le développement du mâle et de la femelle au-dessus d'une supposée température de base de 5°. Le nombre total de degrés-jours de l'oeuf à l'adulte est respectivement de 686 et 706 pour le mâle et la femelle.Les enregistrements des températures maxima et minima dans un abri météorologique à 1 m audessus du sol peuvent être utilisés pour établir le nombre de degrés-jours accumulés du 1 er'mai au 12 juin; à partir de ces données les pics d'émergence de D. coarctata peuvent être prédits. Si 350 degrésjours ou plus sont accumulés durant cette période, le pic d'émergence apparaît vers le 20 juin, mais si 200 degres-jours au moins sont notés le pic sera proche du 11 juillet.L'importance des dégâts occasionnés au bleî d'hiver en Angleterre par les larves de D. coarctata en 1953, 1954, 1965 et 1966 dépend largement du nombre d'oeufs pondus, de la date du semis et de la température accumulée en automne et en hiver; au cours de chacune de ces années les cultures à semis tardif auraient eu peu de possibilité de dépasser le stade sensible avant le moment où elles ont été attaquées.

     

HIV-protease inhibitors induce expression of suppressor of cytokine signaling-1 in insulin-sensitive tissues and promote insulin resistance and type 2 diabetes mellitus

Insulin resistance, hyperglycemia, and type 2 diabetes are among the sequelae of metabolic syndromes that occur in 60-80% of human immunodeficiency virus (HIV)-positive patients treated with HIV-protease inhibitors (PIs). Studies to elucidate the molecular mechanism(s) contributing to these changes, however, have mainly focused on acute, in vitro actions of PIs. Here, we examined the chronic (7 wk) in vivo effects of the PI indinavir (IDV) in male Zucker diabetic fatty (fa/fa) (ZDF) rats. IDV exposure accelerated the diabetic state and dramatically exacerbated hyperglycemia and oral glucose intolerance in the ZDF rats, compared with vehicle-treated ZDF rats. Oligonucleotide gene array analyses revealed upregulation of suppressor of cytokine signaling-1 (SOCS-1) expression in insulin-sensitive tissues of IDV rats. SOCS-1 is a known inducer of insulin resistance and diabetes, and immunoblotting analyses revealed increases in SOCS-1 protein expression in adipose, skeletal muscle, and liver tissues of IDV-administered ZDF rats. This was associated with increases in the upstream regulator TNF-alpha and downstream effector sterol regulatory element-binding protein-1 and a decrease in IRS-2. IDV and other PIs currently in clinical use induced the SOCS-1 signaling cascade also in L6 myotubes and 3T3-L1 adipocytes exposed acutely to PIs under normal culturing conditions and in tissues from Zucker wild-type lean control rats administered PIs for 3 wk, suggesting an effect of these drugs even in the absence of background hyperglycemia/hyperlipidemia. Our findings therefore indicate that induction of the SOCS-1 signaling cascade by PIs could be an important contributing factor in the development of metabolic dysregulation associated with long-term exposures to HIV-PIs.     

Standard quantitative assays for apoptosis

The term apoptosis refers to a peculiar morphology of cell death. It is of special interest because it can be triggered physiologically (and pathologically), and it is regulated by the actions of specific gene products. Therefore, it can in principle be activated and suppressed by medical intervention. It thus is often important to determine whether cells are dying by apoptosis (or its less regulated counterpart, necrosis) and also to quantity the effect in a population of cells. Here the classic methods of apoptosis quantitation are described; they will be of particular use to those whose laboratories are set up for standard microscopical and biochemical techniques, who do apoptosis assays infrequently but wish them to be widely accepted and reproducible. A simple microscopic observation, using blue light illumination and a pair of fluorescent dyes, is recommended for most applications.     

Analysis of differentiation antigens on normal and carcinogen-altered rat epithelial cells in vivo and in culture

Two murine monoclonal antibodies, 3BG8 and 9BG8, which were raised against a rat tracheal squamous-cell-carcinoma cell line, recognize cell-surface antigens on normal rat squamous epithelium (skin, esophagus, vagina, and cornea) as well as on carcinogen-exposed, immortalized, rat tracheal epithelial cells. Monoclonal antibody 3BG8 binds to a 115-kilodalton cell-surface protein on undifferentiated basal cells of the epithelium, while the binding of the other antibody, 9BG8, occurs in both differentiated and undifferentiated populations of normal squamous epithelium and squamous cell carcinomas. Undifferentiated tracheal carcinomas bound only the 3BG8 antibody. No binding of either antibody was detected on normal tracheal mucociliary epithelium. Only under conditions that induce squamous differentiation of rat tracheal epithelium was binding of 3BG8 and 9BG8 detected. For reasons which are not clear at present, 9BG8 dramatically inhibits the growth of normal tracheal and esophageal cells in primary culture, whereas only 3BG8 affects the growth of carcinogen-altered tracheal cell lines. Based on antigen characterization and distribution, it is concluded that the 3BG8 and 9BG8 epitopes are localized on differentiation antigens which differ from others that have been previously described.