Gcn5p, a transcription-related histone acetyltransferase, acetylates nucleosomes and folded nucleosomal arrays in the absence of other protein subunits

Gcn5p is the catalytic subunit of several type A histone acetyltransferases (HATs). Previous studies performed under a limited range of solution conditions have found that nucleosome core particles and nucleosomal arrays can be acetylated by Gcn5p only when it is complexed with other proteins, e.g. Gcn5-Ada, HAT-A2, and SAGA. Here we demonstrate that when assayed in buffer containing optimum concentrations of either NaCl or MgCl2, purified yeast recombinant Gcn5p (rGcn5p) efficiently acetylates both nucleosome core particles and nucleosomal arrays. Furthermore, under conditions where nucleosomal arrays are extensively folded, rGcn5p acetylates folded arrays approximately 40% faster than nucleosome core particles. Finally, rGcn5p polyacetylates the N termini of free histone H3 but only monoacetylates H3 in nucleosomes and nucleosomal arrays. These results demonstrate both that rGcn5p in and of itself is catalytically active when assayed under optimal solution conditions and that this enzyme prefers folded nucleosomal arrays as a substrate. They further suggest that the structure of the histone H3 N terminus, and concomitantly the accessibility of the H3 acetylation sites, changes upon assembly into nucleosomes and nucleosomal arrays.     

Time-dependent changes in heart rate and pupil size during desflurane or sevoflurane anesthesia

To better characterize alterations in autonomic function associated with prolonged anesthesia, we tested the hypothesis that the time-dependent effects of sevoflurane and desflurane differ. We studied seven male volunteers, each anesthetized for 8 h with 1.25 minimum alveolar anesthetic concentration desflurane on one study day and with 8 h sevoflurane on another. These volunteers did not undergo surgery and were minimally stimulated during the study. Measurements included blood pressure, heart rate, pupillary size and light reactivity, concentrations of serum catecholamines, and carbon dioxide production. Over time, heart rate and pupil size increased significantly. During 6 of the 14 anesthetics (45%), heart rate at some point exceeded 95 bpm; similarly, pupil size at some time exceeded 5 mm during 8 anesthetics (57%). In contrast, plasma catecholamine concentrations and carbon dioxide production remained unchanged, and blood pressure remained nearly constant. There are thus substantial time-dependent changes in autonomic functions during prolonged anesthesia, even in unstimulated, nonsurgical volunteers, but we could not detect a difference in these changes during desflurane compared with sevoflurane anesthesia. Implications: Pupil size and heart rate changes are used to guide the delivery of anesthesia. In volunteers, pupil size and heart rate increased with increasing duration of constant desflurane or sevoflurane anesthesia. Thus, anesthetic duration alters heart rate and pupil size independent of surgery and changes in anesthetic delivery.     

Oral malodor in beagles: association with indicators of periodontal disease

The study of oral malodor continues to receive attention. Most bad breath is of oral origin and can be corrected with proper oral hygiene. Studies performed with saliva from people with periodontal disease and from healthy individuals showed that saliva from diseased patients produced a more objectionable odor faster than that of healthy people, and that the volatile sulfur components (VSC) produced may actually play a role in the etiology of periodontal disease. However, not all people or animals with bad breath have periodontal disease. The objectives of this study were to determine if a trained panel could discriminate between 10 dogs with clinically defined periodontal disease and those with relatively healthy periodontium. Second, this study attempted to establish a correlation between odor intensity and six clinical parameters of oral health. The judges were able to differentiate between the two groups of dogs based only on oral malodor (p < 0.02). There were strong associations of the intensity of oral malodor with oral health index scores. The correlations established between odor and gingivitis (r = 0.81) and between odor and furcation exposure (r = 0.88) were very high and statistically significant. Similarly, probing depth (r = 0.73), plaque (r = 0.07) and tooth mobility (r = 0.66) showed clear, positive relationships with oral malodor.     

Immunocytochemical localization of dopamine and its synthetic enzymes in the central nervous system of the lamprey Lampetra fluviatilis

The distribution of dopamine (DA)-containing cell bodies, fibers, and terminals in the brain and spinal cord of Lampetra fluviatilis was investigated by immunohistochemical means. In order to distinguish dopaminergic neurons from those using other catecholamines as the primary neurotransmitter, the distribution of dopamine-immunoreactive structures was compared to that of cell bodies, fibers, and terminals labelled with antibodies directed against the enzymes tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DBH), and phenylethanolamine-N-methyl transferase (PNMT). We define dopaminergic neurons as those that are simultaneously DA, TH, and AADC immunoreactive and at the same time DBH and PNMT nonreactive. The overall concentrations of dopamine, noradrenaline, and adrenaline and some of their metabolites were also measured via high-performance liquid chromatography of whole-brain extracts. Our results revealed the presence of 10 populations of dopaminergic neurons in the brain of the lamprey in the olfactory bulb, preoptic area, hypothalamus, rhombencephalon, and spinal cord. In addition, uniquely DA-immunoreactive neurons, in contact with the cerebrospinal fluid, were observed in the hypothalamus and spinal cord. Chromatography indicated that dopamine exists in considerably higher concentrations than noradrenaline in the lamprey brain, whereas adrenaline is absent, the latter finding being supported by our failure to observe any PNMT-immunoreactive cell bodies, fibers, or terminals. The dopaminergic system of the lamprey appears to share many features not only with that of other anamniotes but also with that of amniotes; however, as in teleosts, dopaminergic neurons in the midbrain corresponding to the substantia nigra, the retrorubral area, and the ventral tegmental area of other species do not exist in the lamprey.     

Persistence of hepatitis C virus in newborn mice brain cell culture

A model of chronic infection of primary cultures of suckling mouse brain (SMB) cells actively producing hepatitis C virus (HCV) is developed. Destruction and repopulation of cells was observed for at least 6 months; this phenomenon was paralleled by virus release in culture medium. Persistent HCV contained in SMB cultures induced a cytopathogenic effect in PS, BHK-21, Vero, HAK, and click embryo cell cultures, its infective titers being 10.0-12.0 lg TCD50/0.2 ml. Persistent HCV formed heterogeneous plaques under agar in chick embryo cells. The polymerase chain reaction (PCR) regularly detected the HCV RNA at the stage of cell destruction in the culture fluid of HCV-infected cell cultures. The cytopathogenic activity of persistent HCV was neutralized by anti-HCV positive patients' sera with the neutralization index of 8.0-9.0 lg. The results of persistent HCV neutralization were confirmed by PCR. Immunofluorescence detected virus-specific HCV antigens in 15-40% of infected cells. Hence, the SMB-HCV system realized the cytopathogenic potential of HCV circulating in the blood of patients with hepatitis C. This system is promising for the study of the pathogenesis of HCV infection at a cellular level, for screening for specific and nonspecific antiviral agents, and for preparing native virus-specific proteins and RNA.     

Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3

P-selectin expressed on the surface of endothelium mediates leukocyte adhesion in vitro and rolling in vivo. Several inducers of cell-surface P-selectin expression on endothelial cells (EC) have previously been identified, all of which yield transient cell-surface expression of P-selectin lasting minutes to a few hours. We now show that a T-lymphocyte product, interleukin-3 (IL-3), stimulates the long-term endothelial cells (HUVEC). IL-3 induced cell-surface P-selectin expression in two phases. An initial peak at 10 minutes was followed by a prolonged upregulation beginning 16 hours after IL-3 addition and lasting at least 4 days. The level of P-selectin expression induced by IL-3 added for 48 hours was similar to that induced by treatment of HUVEC for 10 minutes with thrombin, and the effect of adding IL-3 for 48 hours followed by thrombin for 10 minutes was additive. Induction of cell-surface P-selectin expression by IL-3 was blocked by pretreatment of EC with a blocking monoclonal antibody against the IL-3 receptor alpha-chain. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) and a mutant form of IL-3 with decreased potency did not induce cell-surface P-selectin expression after 48 hours' incubation with HUVEC, suggesting that the effect was specific to IL-3. The increase in cell-surface P-selectin expression occurring after 16 hours of incubation with IL-3 was accompanied by a similar prolonged increase in the steady-state mRNA level that was not observed at 10 minutes after IL-3 addition. As T-lymphocyte infiltration is a hallmark of chronic inflammation, our observations suggest that the secretion of IL-3 by T lymphocytes may serve to maintain the inflammatory state during chronic inflammation.     

Lethal infection of newborn mice, caused by hepatitis C virus

Intracerebral infection of mice aged 2-3 days with hepatitis C virus (HCV) from chronically infected suckling mouse brain cultures and porcine embryo cells resulted in the death of animals after 12-14 days. Repeated passages of HCV in animals decreased the incubation period to just 3-4 days. Blood serum of surviving mice collected during the 4th week postinfection contained antiHCV antibodies detected by enzyme immunoassay and hemagglutination inhibition test. The results are proof of creation of a new in vivo model of experimental HCV infection.