Serial passage of MC3T3-E1 cells down-regulates proliferation during osteogenesis in vitro.

Generally, fibroblast-like cells and other types of human cells have been used to demonstrate the principles of replicative senescence in vitro and in vivo. These cells go through three stages of proliferation, including vigorous proliferation, declining proliferation and quiescence or no proliferation. Any variation of this process occurring in osteoprogenitor cells may offer insight into the mechanism of age-related osteopaenia that predisposes individuals to osteoporosis and bone fractures. We selected MC3T3-E1 cells derived from mouse calvaria to study the mechanism of replicative senescence of pre-osteogenic cells because: (i) these cells constitute a well-known model for studying osteogenesis in vitro; (ii) they undergo a developmental sequence of proliferation and differentiation similar to primary cells in culture; and (iii) they show signs of replicative senescence. These cells were aged by multiple passaging before their use for studying growth kinetics and the effects of population density, effect of extracellular matrix (ECM), size and phases of the cell cycle. Our results show that (i) MC3T3-E1 cells go through the first two stages of proliferation in a manner similar to human cells, but escape the quiescent phase; (ii) the rate of proliferation is similar for low passage (LP) and high passage (HP) cells, but is decreased in very high passage cells (VHP); (iii) growth inhibition is observed using HP cells seeded at high density; (iv) HP ECM stimulates proliferation of both LP and HP cells; (v) a small increase in cell size is observed in HP cells, but no change is seen in the distribution analysis of their cell cycle; (vi) distribution analysis of the cell cycle of VHP cells reveals a decreased and an increased frequency of cells in S and G2 + M phases of their cell cycle, respectively. These results suggest that the mouse MC3T3-E1 cell line exhibits many of the cellular and molecular markers associated with replicative senescence in culture as defined by human cells, such as fibroblast-like cells. Alteration in the sensitivity of MC3T3-E1 cells to intercellular contact and increase in cell size are the primary factors contributing to decreased proliferation of HP cells.

Extracellular matrix alters the relationship between tritiated thymidine incorporation and proliferation of MC3T3-E1 cells during osteogenesis in vitro.

Bone cells in vivo exist in direct contact with extracellular matrix, which regulates their basic biological processes including metabolism, development, growth and differentiation. Thus, the in vitro activity of cells cultured on tissue culture treated plastic could be different from the activity of cells cultured on their natural substrate. We selected MC3T3-E1 pre-osteoblastic cells to study the effect of extracellular matrix on cell proliferation because these cells undergo a progressive developmental sequence of proliferation and differentiation. MC3T3-E1 cells were cultured on plastic or plastic coated with ECM, fibronectin, collagen type I, BSA or poly l-lysine and their ability to proliferate was assessed by incorporation of [3H]dT or by enumeration of cells. Our results show that (1) ECM inhibits incorporation of [3H]dT by MC3T3-E1 cells; (2) collagen type I, but not BSA, poly l-lysine or fibronectin also inhibits incorporation of [3H]dT; (3) the level of ECM inhibition of [3H]dT incorporation is directly related to the number of cells cultured, but unrelated to the cell cycle distribution or endogenous thymidine content; (4) the kinetic profile of [3H]dT uptake suggest that ECM inhibits transport of [3H]dT from the extracellular medium, and (5) cell counts are similar in cultures whether cells are grown on plastic or ECM. These results suggest that decreased incorporation of [3H]dT by cells cultured on ECM is not reflective of bone cell proliferation.

Electroencephalographic changes induced by a noise generator in tinnitus patients and healthy controls.

The present study was conducted in an attempt to determine whether noise generators (NGs) induce changes of electroencephalographic activity in healthy control subjects and in subjects suffering from tinnitus. The results indicated that the application of an NG, irrespective of its placement, induced a significant increase of the average total power in both the female and male tinnitus groups. However, no significant changes were observed for the male control group. A weak increase of average total power was noted for the female control group with the NG placed either in the left or the right ear. The NG induced significant changes of average power in different frequency bands. In conclusion, the NG-induced electroencephalographic changes were dependent on gender, tinnitus location, and placement of the NG.

Electroencephalography correlates in tinnitus.

This study was conducted in an attempt to determine whether the quantitative electroencephalograph activity differs between normal control subjects and subjects suffering from tinnitus. Results indicated that male tinnitus patients as a group had a significantly reduced average total power as compared to control subjects. This finding contrasted with female tinnitus patients, who as a group had a higher average total power as compared to normal female control subjects. Topographical maps (control value-tinnitus value) indicate that with male patients, the frontocentral regions of the brain show the greatest difference. For the female tinnitus patients, the brain regions most affected are the central, parietal, temporal, and occipital regions.

Quantitative electroencephalography and tinnitus: a case study.

We report changes in quantitative electroencephalography activity in a male tinnitus patient when his tinnitus suddenly disappeared. Topographical illustration of the quantitative electroencephalography data showed beta foci in T3 and C4 with tinnitus, which resolved on spontaneous remission of the tinnitus. Comparison of the power spectra in the presence and absence of tinnitus revealed significant changes of a 16-Hz band. Also, a significant increase in alpha power was observed after remission of the tinnitus.

Hemispheric asymmetry in depression and mania. A longitudinal QEEG study in bipolar disorder.

BACKGROUND: previous research has been inconclusive about the nature of hemispheric asymmetry in emotional processing. METHOD: 13 patients with DSM-IV bipolar disorder received repeated QEEGs over 2 years in different mood states. Z-score measures of asymmetry were assessed. RESULTS: asymmetry in frontotemporal slow-wave activity appeared to be in opposite directions in depression compared to mania/hypomania. CONCLUSIONS: mood change in bipolar disorder is associated with change in QEEG asymmetry. LIMITATIONS: study of larger numbers of more homogenous patients under similar conditions is needed. CLINICAL RELEVANCE: study of mood state-dependent asymmetry changes in bipolar disorder may lead to better understanding of hemispheric processing of emotion.

Acyl-coenzyme A causes Ca2+ release in pancreatic acinar cells.

The regulation of cytosolic Ca2+ is important for a variety of cell functions. One non-inositol 1,4,5-trisphosphate (IP3) compound that may regulate Ca2+ is palmitoyl-coenzyme A (CoA), a fatty acid-CoA that is reported to cause Ca2+ release from intracellular stores of oocytes, myocytes, and hepatocytes. To study the role of palmitoyl-CoA in the pancreatic acinar cell, rat pancreatic acini were isolated by collagenase digestion, permeablized with streptolysin O, and the release of Ca2+ from internal stores was measured with fura-2. Palmitoyl-CoA released Ca2+ from internal stores (EC50 = 14 microM). The palmitoyl-CoA-sensitive pool was distinct from, and overlapping with the IP3-sensitive Ca2+ pool. The effects of submaximal doses of IP3 or cyclic ADP-ribose plus palmitoyl-CoA were additive. Fatty acid-CoA derivatives with carbon chain lengths of 16-18 were the most potent and efficacious. Ryanodine and caffeine or elevated resting [Ca2+] sensitized the Ca2+ pool to the actions of palmitoyl-CoA. Fatty acid-CoA levels in pancreatic acini were measured by extraction with 2-propanol/acetonitrile, followed by separation and quantification using reverse phase high performance liquid chromatography, and were found to be 10.17 +/- 0.93 nmol/mg protein. These data suggest the presence of an IP3-insensitive palmitoyl-CoA-sensitive Ca2+ store in pancreatic acinar cells and suggest that palmitoyl-CoA may be needed for Ca2+-induced Ca2+ release.

Serum alpha 1-antichymotrypsin level as a marker for Alzheimer-type dementia.

Excessive alpha 1-antichymotrypsin (ACT) in brain has been postulated to play a role in the pathogenesis of Alzheimer's disease (AD). We measured serum ACT by radial immunodiffusion in 57 patients with presumed AD, 110 healthy controls (24 children; 86 adults), 67 non-AD patients from a geriatric private practice and a VA nursing home, and 136 asthmatics (56 adults; 80 children) as an inflammatory disease control group. Serum ACT was significantly higher in AD (73.1 +/- 22 mg/dl) than in healthy controls (47.9 +/- 8.1 mg/dl) or non-AD patients (61.8 +/- 23.9 mg/dl). A level of 60 mg/dl best separated AD patients from controls or non-AD patients. Serial measurements served to distinguish elevations of ACT level in AD from non-AD inflammatory conditions; the ACT level in the latter returned to normal with therapy or time, but the levels in AD remained elevated. A measure of serum ACT by radial immunodiffusion can be used to support a diagnosis of AD disease but not necessarily as a screening test due to the potentially large number of false positives (26% in the population studied) should malignancy or inflammatory disease be concurrent.

Electroencephalographic cartography of conscious states.

We analyzed monopolar recordings obtained from a brain area implicated in the implementation of conscious intention--the Supplementary Motor Area. Other monopolar recordings were taken at F3, F4, C3, C4, P3, P4, O1, O2, F7, F8, T3, T4, T5, T6, and Cz using the standard international (10/20) pattern with linked ears as reference. Using on line fast-fourier transforms of brainwave signals and computer displays with one-second updates of 1Hz wide brainwave bands of 5, 7, 10, 12, 14, 16, 20 and 28 Hz, specific brainwave signatures were identified for attentional, cognitive, imaginal, and somatosensory states. This first, and thus limited, cartography of consciousness is discussed in light of the cognitive descriptions clinically ascribed to the commonly used brainwave frequency bands designated as Delta, Theta, Alpha, and Beta.

Neurochemical correlates of the Klüver-Bucy syndrome by in vivo microdialysis in monkey.

The Klüver-Bucy syndrome is a well known consequence of lesions of the temporal lobe, but the neural mechanisms remain obscure. To elucidate the neurochemical changes in this syndrome, we utilized in vivo microdialysis of amygdala and hypothalamus in two Cebus monkeys (C. apella) before and after bilateral lesions of the temporal pole (TP). Both subjects were housed and observed in a social group when not being dialyzed. Behavioral changes consequent to the TP lesion included early postoperative anorexia, adipsia, hunched posture, tameness, and lethargy. Subsequently loss of fear, hyperorality, loss of social rank, and social withdrawal were observed. Neurochemical changes in amygdala included fall in DA metabolites, increase in NE, and fall in 5-HIAA. The amino acids glutamate and aspartate were both lower postoperatively but more so in the subject with the greatest behavioral changes. Similar changes were noted in hypothalamus except for DA metabolites which remained unchanged. The Klüver-Bucy syndrome consequent to ablation of the temporal pole appears related to a partial deafferentation of excitatory projections to amygdala, along with a lowering of DA and 5-HIAA and an increase in NE.

Autonomic and skeletal muscle responses to nonelectrical cutaneous stimulation.

Cutaneous stimulation has had a long history as a method of pain control. While there is general agreement that modern techniques such as electrical stimulation and massage often provide relief from acute pain and may in some cases significantly affect chronic pain, the mechanism by which these techniques affect pain remains unclear. Significant attention has been focused on the effects of stimulation on the autonomic nervous system (ANS) along with the increasing evidence of important ANS modulation of nociceptive activity throughout the pain pathway. However, inconsistent results on the presence and direction of ANS changes from cutaneous stimulation characterize the recent literature. The present study investigated a nonelectrical cutaneous stimulation device, the Dermapoints Massageroller, as well as an active placebo massage. The results indicate that the Dermapoints Massageroller has both general effects associated with simple skin stimulation (such as increased skin temperature), as well as specific effects from increased stimulation by the toothed design of the roller. These specific effects include decreased muscle tension (at least for some muscle sites) and increased sympathetic activation. The results are consistent with a model of activation of Pacinian receptors as a possible mechanism for the antinociceptive properties of cutaneous stimulation.

Effect of menses, estrogens and hemolysis on a serum-lectin-like factor in cystic fibrosis.

Twelve CF heterozygous and two CF homozygous women who were tested serially for the CF-lectin activities during one to two months of their menstrual cycles, were found consistently to have negative tests during menses. The specific hormonal alteration during menses that affects the assay is unknown. However, estrogenic medication administered without progesterone to postmenopausal heterozygous women also caused false-negative tests, suggesting that a balance between progesterone and estrogen is critical for the CF-lectin activity; mere depletion of these hormones alone does not interfere. The addition of estrogen in vitro also inhibited the CF-lectin activity at a physiologic concentration of 10(-6) M, and addition of an equivalent concentration of progesterone blocked this inhibitory effect. Future studies of the CF-lectin or its assay as a potential CF-carrier test must be limited to those women not menstruating at the time of blood drawing and not receiving estrogens. Hemolysis or contamination of the serum with RBC should invalidate that sample for testing, although the effect of RBC contamination can be circumvented with the addition of mannose. A more objective, simplified means of performing the CF-lectin assay is reported. A preliminary blind study taking these issues into consideration was 100 percent correct in detecting 16 CF patients and 17 obligate heterozygotes, and revealed 4 percent of 64 control subjects to have positive tests. The presence of this lectin-like factor may reflect the underlying biochemical defect responsible for this disease.

Tacrine in the treatment of Alzheimer's disease. A clinical update and recent pharmacologic studies.

The initial report of the effectiveness of tacrine or tetrahydroaminoacridine (THA) in the treatment of some patients with Alzheimer's disease has been confirmed by further study of additional subjects and by preliminary reports from other investigators. The major side effect, elevation of liver enzymes, is shown to be reversible, dose-dependent and without significant hepatic pathology. Therapeutic serum concentrations of THA vary between 7 and 20 ng/ml. In addition to its presumed action as an acetylcholinesterase inhibitor, data are presented demonstrating marked effects on other brain neurotransmitters in animal models.

Oral tacrine administration in middle-aged monkeys: effects on discrimination learning.

We studied the effect of chronic, oral administration of 1,2,3,4 tetrahydro-9-aminoacridine (THA), an anticholinesterase, on the acquisition of a color discrimination task in five monkeys (Macaca radiata), aged 13-19 years. A two-phase experiment was performed: initially, one animal was used and served as his own control in a multiple dose, crossover, placebo controlled trial, designed to establish a dose-response curve and an optimal dose range based on THA serum concentrations. Thereafter, four monkeys were given the optimal dose of THA (5.0 mg/day) determined previously while learning up to four color pair discriminations. They also learned up to four other color pair discriminations while on placebo. Two monkeys received THA first, then placebo; the others received placebo first, then THA. No order effects were noted. When combined scores for THA tests were compared to their placebo scores, the difference was significant at p less than 0.01 with all four THA treated monkeys requiring fewer trials to reach learning criterion. These results indicate that THA has a significantly positive effect on the acquisition of a color discrimination task.

Long-term oral administration of memory-enhancing doses of tacrine in mice: a study of potential toxicity and side effects.

Recently, tacrine (1, 2, 3, 4-tetrahydro-9-aminoacridine; THA; TAC) has received international attention as an oral agent capable of relieving some of the cognitive symptoms accompanying Alzheimer's disease (AD). When given acutely and parenterally (by injection), tacrine has also enhanced memory retention in animals and man. This study evaluates the clinical potential of this agent by assessing toxicity and major side effects of a memory-enhancing dose of tacrine in mice. Groups of mice received either tacrine or vehicle (placebo) orally for 4 to 6 months. A lack of toxicity after this prolonged treatment with TAC was indicated by: (a) no significant impairment on a battery of behavioral toxicity tests; (b) improved memory retention; (c) a significant but only slight elevation of ornithine transcarbamylase activity in blood serum; (d) no abnormality as revealed with light microscopy of liver tissue; and (e) no gross organ pathology in visceral organs.

Isolation and the fluorometric, high-performance liquid chromatographic determination of tacrine.

Tacrine (THA; 1,2,3,4-tetrahydro-9-aminoacridine) is an anticholinesterase agent which has been used clinically, most recently in the treatment of Alzheimer-type dementias. This paper describes the methodology for the isolation and quantitation of THA at therapeutic levels in serum from human subjects. Using C18 Bond Elut columns and an HPLC/fluorometry system, this assay exhibits a considerable improvement in sensitivity over previous uv methods, and allows routine testing of THA levels in serum samples of reasonable volume from human subjects.

Oral tetrahydroaminoacridine in long-term treatment of senile dementia, Alzheimer type.

We treated 17 patients who had moderate to severe Alzheimer's disease with oral tetrahydroaminoacridine (THA), a centrally active anticholinesterase, in a three-phase study. In the nonblinded first phase of the study, significant improvement occurred in subjects who received the drug, as compared with their pretreatment status, on the global assessment (P = 0.001), the Orientation Test (P = 0.001), and the more sophisticated Names Learning Test (P = 0.001). During the second phase, the subjects served as their own controls in a double-blind, placebo-controlled, cross-over study in which the order of administration of the drug and placebo was randomly assigned. Among the 14 subjects completing Phase II, THA treatment produced significantly better results than placebo on the global assessment (P = 0.003), the Orientation Test (P = 0.004), the Alzheimer's Deficit Scale (P = 0.003), and the Names Learning Test (P = 0.001). Twelve subjects have entered Phase III, which involves long-term administration of oral THA. The average duration of treatment in these subjects at present is 12.6 months; symptomatic improvements have occurred, and no serious side effects attributable to THA have been observed. These encouraging initial results suggest that THA may be at least temporarily useful in the long-term palliative treatment of patients with Alzheimer's disease. We stress that further observations will be required before a clear assessment of the role of this agent can be made.

Abnormal feeding behavior of western toads (Bufo boreas) kept in a hyperosmotic environment. I. A quantitative behavioral analysis as related to cerebral amino acid concentrations.

Discriminant analysis of eleven behavioral variables associated with feeding permitted the assignment of hyperosmotically-acclimating (HOA) toads (Bufo boreas) to six different behavioral states. These behavioral states could be correlated with specific alterations in the level of select amino acids in three regions of the toad's central nervous system. By considering only those amino acids that showed equivalent levels in corresponding brain regions of normally-behaving, freshwater-acclimating (FWA) and HOA toads, it was possible to focus upon just nine amino acids as possible modulators of feeding behavior. Four of these amino acids were markedly elevated in two or more abnormal behavioral states: gamma-aminobutyric acid (GABA) in all three brain areas; glutamate in the cerebral hemispheres; aspartate in the cerebral hemispheres and olfactory bulbs; and phenylalanine in the olfactory bulbs and optic lobes. Other possible behavior modulators identified were: lysine and threonine in the cerebral hemispheres; carnosine in the olfactory bulbs; and valine and alanine in the optic lobes.