Induction of a cellular enzyme for energy metabolism by transforming domains of adenovirus E1a.

Brain creatine kinase is a major enzyme of cellular energy metabolism. It is overexpressed in a wide range of tumor cell lines and is used as a tumor marker. We reported recently that the promoter of the human gene has a strong sequence similarity to the adenovirus E2E promoter. This similarity suggested that the brain creatine kinase gene may be regulated by the viral activator E1a. Experiments reported here showed that both enzyme activity and mRNA levels were induced by the oncogenic products of the E1a region of adenovirus type 5, but unlike the viral E2E promoter, which is induced predominantly by E1a domain 3, brain creatine kinase induction required domains 1 and 2. These domains are important for transformation and for the association of E1a with the retinoblastoma gene product and other cellular proteins. The induction by an oncogene of a cellular gene for energy metabolism may be of significance for the metabolic events that take place after oncogenic activation.     

The role of evolutionary intermediates in the host adaptation of canine parvovirus

The adaptation of viruses to new hosts is a poorly understood process likely involving a variety of viral structures and functions that allow efficient replication and spread. Canine parvovirus (CPV) emerged in the late 1970s as a host-range variant of a virus related to feline panleukopenia virus (FPV). Within a few years of its emergence in dogs, there was a worldwide replacement of the initial virus strain (CPV type 2) by a variant (CPV type 2a) characterized by four amino acid differences in the capsid protein. However, the evolutionary processes that underlie the acquisition of these four mutations, as well as their effects on viral fitness, both singly and in combination, are still uncertain. Using a comprehensive experimental analysis of multiple intermediate mutational combinations, we show that these four capsid mutations act in concert to alter antigenicity, cell receptor binding, and relative in vitro growth in feline cells. Hence, host adaptation involved complex interactions among both surface-exposed and buried capsid mutations that together altered cell infection and immune escape properties of the viruses. Notably, most intermediate viral genotypes containing different combinations of the four key amino acids possessed markedly lower fitness than the wild-type viruses.     

Acid Orcein-Iron and Acid Orcein-Copper Stains for Elastin

Paraffin sections of formol-fixed tissues stained 4-18 hr in 70% alcohol containing 1% orcein and 1% of concentrated (12 N) HCl by volume yield the familiar purple brown elastin and red nuclei on a pink background. When sections so stained are transferred directly from the stain to 70% alcohol containing 0.02% ferric chloride (FeCl₃·6 H₂O) or 0.02% copper sulfate (CuSO₄·5 H₂O) for a 15 sec to 3 min period, elastin coloration is changed to black or reddish black and chromatin staining to reddish black. The procedure can be counterstained with picro-methyl blue to yield blue collagen and reticulum or with our flavianic acid, ferric chloride, acid fuchsin mixture to give deep yellow background and deep red collagen.     

Variation in the transition from inside to outside work in the red harvester ant Pogonomyrmex barbatus

Summary. In laboratory colonies of the red harvester ant, Pogonomyrmex barbatus, we observed the sequence of tasks performed by marked individuals. Observations of about 760 ants in three laboratory colonies indicate that ants often move from inside to outside work. However, there was a great deal of variation in the sequence. If trends in sequence were weak because ants do move from inside to outside work but the duration of our observations was too short to see the transition, ants should be observed to stay either inside or outside. There was no significant tendency for ants to persist in inside or outside work, indicating the variability in sequence is real. Ants tended to perform midden work before they died. Foraging activity is low in laboratory colonies, and it may be that ants that would be foragers in the field end up as midden workers in the laboratory. High variability in task sequence, in uniform laboratory conditions, contrasts with the apparently more consistent sequence from inside to outside work in the field. This suggests that requirements imposed by variable external conditions and colony needs in the field have a strong influence on task sequence.Received 7 May 2004; revised 11 November 2004; accepted 18 November 2004.     

Nucleotides and divalent cations as effectors and modulators of exocytosis in permeabilized rat mast cells.

The idea that the universal trigger to exocytosis (the terminal step in the secretory process) is an elevation of the cytosol concentration of Ca2+, and that it is dependent on ATP, is no longer tenable. Working with streptolysin-O-permeabilized mast cells (and other myeloid cells) we have shown that non-hydrolysable analogues of GTP can stimulate exocytosis after depletion of Ca2+ (i.e. at concentrations below 10(-9) M) and ATP. Such Ca2+- and ATP-independent exocytosis is strongly dependent on the presence of Mg2+, and the requirement for Mg2+ declines as the concentration of Ca2+ is brought up to 10(-7) M. We argue that Ca2+ serves to regulate the binding of guanine nucleotides to GE, a GTP-binding protein that regulates exocytosis through its interaction with CE, a calcium-binding protein which serves as an intracellular pseudo-receptor. The onset of exocytosis, following provision of Ca2+ and guanine nucleotides to the permeabilized cells, is preceded by delays which are sensitive to the order of provision of the two effectors (i.e. Ca2+ and guanine nucleotides), the presence or absence of Mg2+, and the identity of the activating guanine nucleotide. In view of the similarity of these features with the activation kinetics of adenylyl cyclase, we argue that GE behaves as a member of the heterotrimeric class of signal transducing G-proteins such as GS.     

Elimination of precipitates in oil red O fat stain by adding dextrin.

Addition of dextrin in the final 60% isopropanol of the Lillie-Ashburn super-saturated oil red O isopropanol technic moderately intensified the stain and decreased our required staining interval. Precipitates were decreased and the diluted solution remained usable into the second week. A saturated 60% isopropanol oil red O solution contained 33 mg/100 ml. Without dextrin the fresh supersaturated solution contains 40 mg, after 3 days 25 mg. With dextrin the fresh solution contained 130 mg dye, the 10-day-old one 100 mg/100 ml.     

Elimination of Precipitates in Oil Red O Fat Stain by Adding Dextrin

Addition of dextrin in the final 60% isopropanol of the Lillie-Ashburu supersaturated oil red O isopropanol technic moderately intensified the stain and decreased our required staining interval. Precipitates were decreased and the diluted solution remained usable into the second week.

A saturated 60% isopropanol oil red O solution contained 33 mg/100 ml. Without dextrin the fresh supersaturated solution contains 40 mg, after 3 days 25 mg. With dextrin the fresh solution contained 130 mg dye, the 10-day-old one 100 mg/100 ml.     

The mechanism of the ferric ferricyanide reduction reaction

Synopsis The ferric ferricyanide reaction has been generally considered to depend on the reduction of ferricyanide to ferrocyanide and, in the presence of the ferric ion, the consequent production of Prussian Blue. In testing the ability of ferricyanide to prevent the azo-coupling reactions of enterochromaffin cells and of the noradrenaline islets of the adrenal medulla, ferricyanide proved to be an effective oxidant in alkaline solution, but failed to quinonize the two catechols below pH 6. Similar results are obtained in oxidative blocking of cutaneous sulphydryl sites against the ferric ferricyanide reaction and against staining by the mercaptide acid azo-coupling reaction of Lillie & Glenner (1965).Ferric ferricyanide solutions are used at pH 2–2.5 and the ferric ion is an effective quinonizing and thiol-oxidizing agent at this pH level; the ferricyanide ion is not. The ferric ferricyanide reaction depends on the reduction of ferric to ferrous ions in the presence of ferricyanide, with the production of Turnbull's Blue.     

Type 2 Bovine Adenovirus as an Adventitious Contaminant in Primary Bovine Embryonic Kidney Cell Cultures

Three lots of primary bovine embryonic kidney cell cultures obtained from commercial sources were found to contain type 2 bovine adenovirus. These cell cultures, apparently derived from healthy fetuses, required long incubation periods before the virus could be detected.