CaM-kinaseII-dependent commitment to microcystin-induced apoptosis is coupled to cell budding, but not to shrinkage or chromatin hypercondensation.

The protein phosphatase inhibitor microcystin-LR (MC) induced hepatocyte apoptosis mediated by the calcium-calmodulin-dependent multifunctional protein kinase II (CaMKII). CaMKII antagonists were added at various times after MC to define for how long the cells depended on CaMKII activity to be committed to execute the various parameters of death. Shrinkage and nonpolarized budding were reversible and not coupled to commitment. A critical commitment step was observed 15-20 min after MC (0.5 microM) addition. After this, CaMKII inhibitors no longer protected against polarized budding, DNA fragmentation, lost protein synthesis capability, and cell disruption. Commitment to chromatin hypercondensation occurred 40 min after MC addition. In conclusion, irreversible death commitment was coupled to polarized budding, but not to shrinkage or chromatin condensation. Antioxidant prevented chromatin condensation when given after the CaMKII-dependent commitment point, suggesting that CaMKII had mediated the accumulation of a second messenger of reactive oxygen species nature.

Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors.

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.

Effect of linseed supplementation of the gestation and lactation diets of dairy ewes on the growth performance and the intramuscular fatty acid composition of their lambs.

In this study, we investigated the effects of maternal gestation and/or lactation diets supplemented with extruded linseed (rich in 18:3n-3) on growth performance and long-chain polyunsaturated faaty acid (PUFA) accumulation in muscle tissues of suckling lambs. A total of 36 dairy ewes were fed a control diet (CON) and a diet containing linseed (LIN) during the last 8 weeks of gestation and/or the first 4 weeks of lactation. The four dietary treatments consisted of the following gestation/lactation feeding treatments: CON/CON, CON/LIN, LIN/LIN or LIN/CON. The lambs born from ewes fed the aforementioned diets were reared exclusively on milk and were slaughtered at 4 weeks of age. Profiles of ewes' milk fatty acids and that of intramuscular fat (IMF) of leg muscles from lambs were determined. Compared with the CON/CON, LIN/CON offspring tended to grow slower and to have reduced cold carcass weights. Moreover, the LIN supplementation only in the prepartum period (LIN/CON) resulted in greater PUFAn-3 accumulation in the IMF compared with the CON/CON offspring due to increased 20:5n-3 (1.20 v. 0.64 mg/100 mg of total FA), 22:5n-3 (1.91 v. 1.46;) and 22:6n-3 (1.25 v. 0.89) contents, respectively. Compared with the CON/CON diet, providing LIN only during lactation (CON/LIN) caused a greater PUFAn-3 content in the IMF mainly due to a greater 18:3n-3 (1.79 v. 0.75 mg/100 g total FA) concentration. Continuous PUFAn-3 exposure, both via the maternal gestation and lactation diet, had no additive effects on PUFAn-3 accumulation in tissues. The results suggest that linseed, as an 18:3n-3 source, seems to be more efficient in increasing long-chain PUFAn-3 in fetal than in suckling lamb tissues.

Cyclic adenosine monophosphate (cAMP) analogs 8-Cl- and 8-NH2-cAMP induce cell death independently of cAMP kinase-mediated inhibition of the G1/S transition in mammary carcinoma cells (MCF-7).

Human mammary carcinoma cells (MCF-7) were arrested in late G1-phase after treatment with agents (forskolin, interleukin-1 beta 3-isobutyl-1-methylxanthine) that increased the endogenous concentrations of cAMP. The effect of elevated cAMP was mimicked by microinjected catalytic (C alpha) cAMP-dependent protein kinase (cAK) subunit and reversed by the injection of a dominant negative cAK regulatory mutant (RID199). Further evidence that activation of cAK induced growth arrest was provided by the use of pairs of stable cAMP analogs known to synergistically activate isolated cAK isozymes. Furthermore, the effect of cAMP was not potentiated by serine/threonine phosphatase inhibitors that profoundly restricted MCF-7 growth. Some 8-substituted cAMP analogs, e.g. 8-Cl-cAMP and 8-NH2-cAMP, induced cell death rather than reversible inhibition of growth. Their effect was not synergized with complementary cAMP analogs. Furthermore, their potency was decreased rather than increased in the presence of an inhibitor of degradation (3-isobutyl-1-methylxanthine). Finally, their effect could be mimicked by degradation products unable to activate cAK. We concluded that 8-Cl-cAMP (and 8-NH2-cAMP) induced irreversible growth arrest by a mechanism not involving cAK, whereas activation of cAK resulted in a transient and fully reversible inhibition of cell proliferation.

Selective cleavage of 28S rRNA variable regions V3 and V13 in myeloid leukemia cell apoptosis.

Vigorous apoptosis is induced 3-4 hours after activation of cAMP-dependent protein kinase I in the rat myeloid leukemia cell line IPC-81 [J. Cell. Physiol. 146 (1991) 73-80]. We will report a novel feature of apoptosis in these cells: a selective and temporarily ordered cleavage within the two largest 28S ribosomal RNA variable regions (V3 and V13). The cleavage of 28S rRNA coincided with internucleosomal DNA fragmentation and cessation of cellular protein synthesis. The implication of 28S variable regions as targets in apoptosis is a clue to the function of these so far apparently superfluous parts of eukaryotic ribosomes.

A procedure for studying echoic control in verbal behavior.

Male and female college students answered standard questions about the women's liberation movement on three occasions. A set of target words was embedded in the questions, with one set used in Sessions 1 and 3, and a synonymous, but different set used in Session 2. The relative frequencies of usage of a given target word were directly related to whether the questions for that session contained the word. The results supported the hypothesis of echoics as proposed in Skinner's theory of verbal behavior.

The choice of resuspension medium for isolated rat liver nuclei: effects on nuclear morphology and in vitro transcription.

Standard protocols for in vitro transcription assay (nuclear run-off) include 10-40% (v/v) glycerol (of various ionic strength) in the medium used for resuspension/storage of the isolated nuclei. In the present work the morphological and functional properties of nuclei isolated from rat liver have been studied as a function of the content of glycerol, sucrose and inorganic ions (K+ and Mg2+) in the resuspension medium. In contrast to earlier reports, glycerol was found not to be essential to maintain morphological integrity and RNA polymerase activity in frozen/stored nuclei. Nuclear pellets, resuspended and stored in isoosmotic sucrose media, were found to give morphologically intact and transcriptionally active nuclei. Furthermore, these nuclei displayed a higher specific hybridization signal for the differentially expressed genes encoding peroxisomal beta-oxidation enzymes, relative to the total RNA synthesis, than nuclei resuspended and stored in a hyperosmotic glycerol-containing medium. The concentrations of inorganic ions were also found to affect nuclear morphology. Flow cytometry indicated DNA leakage from nuclei at insufficient concentrations of K+ and Mg2+, and high ionic strength favoured aggregation and disintegration of nuclei. Our findings indicate that quantitative results from nuclear run-off experiments should be interpreted with caution until the process of transcription in isolated nuclei is better understood.

Cross-infection risks associated with current procedures for using high-speed dental handpieces.

When a dye solution used to simulate patient material was either injected into high-speed dental handpiece (drill) waterlines or applied to the equipment externally, internal air turbine chambers became contaminated. These chambers served as a reservoir of the material, which was slowly dislodged by air expelled during subsequent handpiece operation and which was diluted by water spray used for cooling the drilling surface. Considering the fact that patient materials could reside in internal parts of the equipment that are not usually disinfected and that the material may be subsequently sprayed into cuts and abrasions in the oral cavity, the common approach to reprocessing handpieces (external wiping in combination with flushing) may pose unacceptably high risks to those individuals treated soon after infected patients. Therefore, unless reliable data on cross-infection frequencies are obtained and prove it unnecessary, heat-treating high-speed handpieces between each patient should be considered an essential component of standard procedures whenever universal precautions are practiced in dentistry.

A stereological study of the ependyma of the mouse spinal cord. With a comparative note on the choroid plexus ependyma.

Applying different stereological techniques, the total ependymal volume in the spinal cord of mice was estimated to be 83 x 10(6) microns cubed, the number of cells to be 163,000 and the mean ependymal cell volume to be 510 microns cubed. Compared to choroid plexus cells in the third ventricle, the ependymal cells in the spinal cord contained a smaller mitochondrial volume (9.8% versus 4.6% of cell volume) and less rough endoplasmic reticulum (2.1% versus 0.4%). These findings indicate that the metabolic activity of the ependyma in the spinal cord is lower than that in the choroid plexus. Compared to liver and exocrine pancreatic cells, ependymal cells in both locations must be considered to have a rather low metabolic activity.

8-Chloro-cAMP induces apoptotic cell death in a human mammary carcinoma cell (MCF-7) line.

8-Cl-cAMP and 8-NH2-cAMP induced MCF-7 cell death. The type(s) of cell death were studied in more detail and compared with the cell death type (apoptosis) induced by okadaic acid, an inhibitor of serine/threonine phosphatases. By morphological criteria dying cells showed loss of cell-cell interactions and microvilli, condensation of nuclear chromatin and segregation of cytoplasmic organelles. By in situ nick end-labelling, using digoxigenin-conjugated dUTP as probe, a large fraction of 8-Cl-cAMP, 8-NH2-cAMP and 8-Cl-adenosine-exposed cells stained positively in the advanced stages of death. In the early phase of chromatin condensation the cells stained negatively. Specific (internucleosomal) DNA fragmentation was not observed. The MCF-7 cell death induced by 8-Cl-cAMP and 8-NH2-cAMP was not mediated by activation of the cAMP kinase since more stable cAMP analogues (8-CPT-cAMP and N6-benzoyl-cAMP) or forskolin failed to induce death. Furthermore, 8-Cl-cAMP action was counteracted by adenosine deaminase and 3-isobutyl-1-methylxanthine, and mimicked by 8-Cl-adenosine, a major metabolite of 8-Cl-cAMP. It is concluded that 8-Cl- and 8-NH2-cAMP can induce morphological and biochemical effects resembling apoptotic cell death in MCF-7 cells through their conversion into potent cytotoxic metabolite(s).

Cyclic adenosine monophosphate acts synergistically with dexamethasone to inhibit the entrance of cultured adult rat hepatocytes into S-phase: with a note on the use of nucleolar and extranucleolar [3H]-thymidine labelling patterns to determine rapid changes in the rate of onset of DNA replication.

Analogs of cyclic adenosine monophosphate (cAMP) (N6benzoyl cAMP and N6monobutyryl cAMP) as well as agents that increased the intracellular level of cAMP (glucagon and isobutylmethylxanthine) inhibited the EGF-stimulated DNA replication of adult rat hepatocytes in primary culture independently of cell density. This inhibition was strongly potentiated by the glucocorticoid dexamethasone. The effect of cAMP (and dexamethasone) was not due to toxicity, because the inhibition was reversible and the cell ultrastructure preserved. cAMP acted by decreasing the rate of transition from G1- to S-phase, the duration of G2- and S-phase of the hepatocyte cell cycle being unaffected. DNA replication started in the extranucleolar compartment of the nucleus and ended in the nucleolar compartment as described earlier for cells grown in the absence of cAMP (O.K. Vintermyr and S.O. Døskeland, J. Cell. Physiol., 1987, 132:12-21). The action of cAMP was very rapid: significant inhibition of the transition was noted 2 hr after the addition of glucagon/IBMX and half-maximal inhibition after 4 hours. The determination of extranucleolarly labelled nuclei in cells pulse-labelled with [3H]thymidine allowed precise analysis of rapid changes in the probability of transition from G1- to S-phase. The extranucleolar labelling index could also be determined in cells continuously exposed to [3H]thymidine.

Elevated cAMP gives short-term inhibition and long-term stimulation of hepatocyte DNA replication: roles of the cAMP-dependent protein kinase subunits.

The study reports the role of the isozyme forms (cA-PKI and cA-PKII) and subunits (R and C) of cAMP-dependent protein kinase in mediating the acute depression of hepatocyte DNA replication by elevated cAMP. Combinations of cAMP analogs preferentially activating cA-PKI or II showed that either isozyme could inhibit DNA replication. The effects of glucagon and cAMP analogs were counteracted by the cAMP antagonist RpcAMPS, implicating the necessity for cA-PK dissociation in cAMP action. The effect of elevated cAMP was mimicked by microinjected C subunit, but not by the RI subunit of cA-PK. Hepatocytes under continuous cAMP challenge more than regained their replicative activity. This tardive stimulatory effect of cAMP was enhanced by insulin and blocked by dexamethasone, and was preceded by downregulation of cA-PK. In conclusion, a burst of cAMP acutely inhibits hepatocyte G1/S transition in late G1 regardless of hormonal state. In the presence of high glucocorticoid/low insulin the inhibition persists. At high insulin/low glucocorticoid the inhibitory phase is followed by a prolonged stimulation of DNA replication. Downregulation of endogenous cA-PK is a mechanism for escape from the inhibitory action of highly elevated cAMP.

Hepatocyte DNA replication is abolished by inhibitors selecting protein phosphatase 2A rather than phosphatase 1.

Primary rat hepatocytes exposed to the phosphoprotein phosphatase (PP) inhibitors microcystin-LR and okadaic acid showed extensive surface protrusions and release of cell fragments, like cells in apoptosis. Microinjected microcystin fully reproduced these effects; the calculated intracellular concentration required for 50% effect being about 1 microM. The effects were counteracted by antagonists of calmodulin or of the multifunctional calmodulin-activated protein kinase II. The DNA replication of the epidermal growth factor-stimulated hepatocytes was nearly completely inhibited by okadaic acid at concentrations below those giving overt morphological effects. However, microcystin did not inhibit the DNA replication. Calmodulin antagonists counteracted the effect of okadaic acid on DNA replication. Microinjection of inhibitor-1 and inhibitor-2 (both directed against PP1) had no effect on DNA replication. Based on the known selectivity of okadaic acid for PP type 2A versus that of type 1, and the lack of such selectivity for microcystin, it is concluded that DNA replication is abolished by moderate inhibition of PP2A. Inhibition of PP1 did not impede DNA replication, suggesting that the two major liver phosphatases may have opposite roles in the regulation of hepatocyte DNA replication.

Comparison of ASHA's immittance guidelines and standard medical diagnostic procedures.

Immittance procedures and medical evaluation based on case history and otoscopy were administered to 115 youngsters from a large pediatric practice. Using ASHA immittance guidelines, a reasonable 73% overall agreement was achieved between immittance and medical recommendations of "pass", "at risk", and "fail/treat". When using medical findings as the criterion, there were three definite cases of under-referral (false negatives) and five over-referrals (false positives) in this sample group.

The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells.

Okadaic acid, a specific and potent inhibitor of protein phosphatases 2A and 1, was tested for its effect on the morphology of a number of cell types: freshly isolated rat hepatocytes in suspension or in primary culture, the human mammary carcinoma cell line MCF-7, the human neuroblastoma cell line SK-N-SH, rat pituitary adenoma GH3 cells, and rat promyelocytic IPC-81 cells. All the cell types reacted within a few hours to okadaic acid in the concentration range 0.1 to 1 microM with profound morphological alterations. Among the changes noted were: condensation of chromatin, shedding of cell contents via surface bleb formation, redistribution and compacting of cytoplasmic organelles, formation of cytoplasmic vacuoles, and hyperconvolution of the nuclear membrane. In some cells nuclear fragmentation was noted. In addition, cells growing as monolayers rounded up and detached from the substratum. The treated cells had no swollen mitochondria and retained the ability to exclude trypan blue until the final stage of dissolution, supporting the hypothesis that the changes were apoptotic rather than necrotic. In hepatocytes the action of okadaic acid was mimicked by another phosphatase inhibitor, microcystin, and was accompanied by shrinkage of the cell volume, as judged by Coulter counter analysis. The action of phosphatase inhibitor was not abolished by protein synthesis inhibitors, Ca(2+)-depleted medium, or phorbol ester. Although hepatocyte DNA replication was very sensitive to inhibition by okadaic acid, DNA fragmentation was less pronounced in response to okadaic acid than other agents inducing the morphological appearance of apoptosis.

Human beta-defensin-1 mRNA is transcribed in tympanic membrane and adjacent auditory canal epithelium.

The external auditory canal is less susceptible to infections than the sensitive middle-ear cavity. Since recent research has provided insight to the production of potent antimicrobial peptides from various surface epithelia, we wanted to investigate whether protection of the external auditory canal in part could be explained by the production of human beta-defensin-1 (HBD-1). This particular peptide is known to be constitutively expressed in various surface epithelia, such as airway, skin, and urogenital tissues. By reverse transcriptase PCR we demonstrate HBD-1 mRNA in the pars tensa and pars flaccida of the tympanic membrane and in the meatal skin. In situ hybridization studies localized the HBD-1 mRNA to the epidermal layer of these tissues. The HBD-1 transcripts were also evident in the sebaceous glands and in hair follicles of the meatal skin. In contrast, HBD-1 mRNA was not detected in the tympanal epithelium of the eardrum. The widespread presence of mRNA encoding for this broad-spectrum antimicrobial peptide in the meatal skin and tympanic membrane suggests that HBD-1 participates in the innate antimicrobial defense of the external auditory canal and middle-ear cavity.

Cross-contamination potential with dental equipment.

Some types of reused dental equipment, especially handpieces and their attachments for drilling and cleaning teeth, might be responsible for cross-contamination if patient material were to lodge temporarily in difficult-to-disinfect internal mechanisms. This possibility is worrisome with respect to transmission of hepatitis B and human immunodeficiency viruses (HBV, HIV). Previous cross-contamination studies have relied on laboratory experiments with bacteria or dye tracers. To assess possible risk more thoroughly, we tested 30 new prophylaxis angles and 12 new high-speed handpieces to see whether they would take up and expel contaminants in laboratory and clinical trials. In treatments of three patients, including two infected with HIV, human-specific DNA (beta-globin, HLA DQ alpha) and HIV proviral DNA were detected inside or coming back from the devices. Similarly, when handpieces were operated in contact with blood pooled from HBV-infected patients, HBV DNA was detected in samples taken from inside the equipment and from their attached air/water hoses. When we used bacteriophage phi X174 as a model virus in laboratory tests, many infective viral particles were recovered from internal mechanisms of handpieces, their connecting air/water hoses, and from water spray expelled when the equipment was reused. We recommend that reused high-speed, air-driven handpieces and prophylaxis angles should be cleaned and heat-treated between patients. Further studies are needed to determine ways of eliminating the risks associated with exhaust hoses and air/water input lines.