Voltage sensitivity of the osteoclast calcium receptor

We demonstrated previously that osteoclasts possess a divalent cation-sensitive "receptor", the Ca2+ receptor. Activation of the Ca2+ receptor by the surrogate cation Ni2+ was shown to elicit an increase in cytosolic [Ca2+] to a peak value followed by an exponential decline. In the present study we examined the influence of surface membrane voltage on the kinetics of Ca2+ receptor inactivation. The K+ ionophore, valinomycin was applied to intercept the declining phase of the cytosolic [Ca2+] transient elicited by application of between 50 microM- and 5 mM-[Ni2+]. This resulted in a sustained elevation of cytosolic [Ca2+] or even a 'hump' followed by a gradual decline. Such a kinetic alteration persisted in a Ca(2+)-free solution, but was abolished in high extracellular [K+] (105 mM). Thus, we demonstrate for the first time to our knowledge, a modulatory effect of membrane potential on the function of the osteoclast Ca2+ receptor.     

The course of geriatric depression with "reversible dementia": a controlled study

OBJECTIVE: The goals of this longitudinal investigation were 1) to study the rate of development of irreversible dementia in elderly depressed patients with a dementia syndrome that subsided after improvement of depression and 2) to compare it with that of depressed, never-demented patients. METHOD: The subjects were 57 elderly patients consecutively hospitalized for major depression. At entry into the study, 23 subjects also met criteria for "reversible dementia," while 34 were without dementia. After a systematic clinical evaluation, the subjects were followed up at approximately yearly intervals for an average of 33.8 months. RESULTS: Irreversible dementia developed significantly more frequently in the depressed group with reversible dementia (43%) than in the group with depression alone (12%). Survival analysis showed that the group with reversible dementia had a 4.69-times higher chance of having developed dementia at follow-up than the patients with depression alone. No clinical characteristics at entry into the study were found to discriminate the subjects who developed irreversible dementia during the follow-up period from those who remained nondemented. CONCLUSIONS: These findings suggest that geriatric depression with reversible dementia is a clinical entity that includes a group of patients with early-stage dementing disorders. Therefore, identification of a reversible dementia syndrome is an indication for a thorough diagnostic workup and frequent follow-ups in order to identify treatable neurological disorders.     

Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying.

The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied the causes of this phenomenon is isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25-30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol-ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.     

The influence of node sequence and extraneous load induced by graphical overviews on hypertext learning

The effects of four hypertext learning environments with a hierarchical graphical overview were studied on the coherence of the node sequence, extraneous load and comprehension. Navigation patterns were influenced by the type of overview provided (i.e., dynamic, static) and whether navigation was restricted (i.e., restricted, non-restricted). It was hypothesised that redundant use of the overview for inducing a high-coherence reading sequence would result in high extraneous load and low comprehension. Coherence was higher in the dynamic than in the static conditions. Coherence was also higher in the restricted than in the non-restricted conditions. Mental effort as a measure of extraneous load was higher at the end than at the beginning of the learning phase, especially in the dynamic restricted and the static non-restricted conditions, although there was no significant interaction. Comprehension was lowest in the dynamic restricted condition and highest in the dynamic non-restricted and static restricted conditions. Low comprehension in the dynamic restricted condition indicates that overviews can become redundant for reading sequence coherence, negatively impacting comprehension. The evidence suggests that severe restriction of navigation paths should be avoided and that continuous use of overviews such as in dynamic overviews may be detrimental to learning.     

Telomerase activity and telomere lengths in various cell lines: changes of telomerase activity can be another method for chemosensitivity evaluation

For the cancer cells which have overcome the second mitotic clock (M2), activated telomerase is essential and used as another marker of immortality. Many trials had been initiated to target telomerase, which is known to be specific to tumors. To determine the best in vitro cell system for testing the efficacy of telomerase inhibitors, we evaluated the telomerase activity of various cancer cell lines and measured their telomere lengths. We also treated some cancer cell lines with adriamycin and measured the changes of telomerase activity. Telomerase activity was evaluated in various cell lines with the TRAP (telomeric repeat amplification protocol) assay. Telomerase activity was calculated and translated into arbitrary units by computer-assisted densitometry with the control of telomerase activity in the 293 control cell line. Also, terminal restriction fragment lengths were measured using Southern blotting. We also measured telomerase activity and telomere lengths in 11 benign breast tumor tissues and 19 paired stomach cancer and normal tissues. Cancer cell lines treated with adriamycin we evaluated for changes of telomerase activity and the cell proliferation by MTT assay and dye exclusion test. Telomerase activity of cell lines was 95.3 24.1 unit with a range of 27.6-129.6 unit, while the telomere lengths of those cell lines were variable from 5.0 to 10.4 kbp with a median of 6 kbp. In 11 cancer cell lines which were not yet firmly established, we could not detect any telomerase activity. Low telomerase activity was detected in only 2 benign tumor tissues of breast with a median telomere length of 8.8 (7-10.5) kbp. Among paired 19 gastric cancer and normal tissues, only 7 cancer tissues showed weak telomerase activity. After adriamycin treatment, telomerase activity in YCC-S-1, YCC-S-3, MCF-7 and MCF-7/ADR was decreased in accordance with the changes of the cell numbers. Telomerase is specific to cancer tissues and is expressed differently from organ to organ. Telomerase activity by TRAP assay could be used as a chemosensitivity assay.     

Treatment of irritable colon with the food additive Appital

Appital is a herbal medicine. The aim was to examine its effect in patients with irritable bowel syndrome (IBS). Other diseases were excluded by physical examination, rectoscopy, blood tests and in patients older than 35 years X-ray of the colon or colonoscopy. The study was designed as a double-blind placebo-controlled trial. The patients were randomized to either Appital or placebo. Following two weeks without medicine, the patient had Appital or placebo for eight weeks. Fifty-nine patients were randomized, 47 completed the study. The results were based on symptom scores registered by the patients. The symptom score was significantly reduced in patients treated with Appital (p = 0.002), but when compared to placebo, the difference was insignificant (p = 0.081). We concluded that Appital has no effect in relieving symptoms of IBS compared to placebo, although due to the possibility of type two error we cannot exclude a small, but hardly clinically relevant effect.     

In vivo heat shock protects rat myocardial mitochondria

Heat shock (HS)/stress proteins (HSP) provide protection from a variety of stresses other than HS, including oxidative stress and mitochondria have been implicated as the target of HS-related protection in stressed cultured cells. Here we investigated whether mitochondria also are targets for the HS-mediated protection in vivo. Sprague Dawley rats were exposed, or not, to HS (41 degrees C, 15 min). After a 21 h recovery period, hearts were excised and perfused with or without H2O2 (0.15 mM). Myocardial mitochondria were then isolated, and their oxygen consumption was analyzed. HS prevented H2O2-induced alterations in state 3 respiration while increasing the expression of Hsp70 and heme oxygenase (HO). Thus, in vivo HS protects rat myocardial mitochondrial respiration against the deleterious effects of oxidative injury, a protection relating to Hsp70 and/or HO and targeting state 3 respiration.     

Effects of vesnarinone, a novel orally active inotropic agent with an immunosuppressive action, on experimental cardiac transplantation in rats

BACKGROUND: Vesnarinone (VES) has been used for treatment of patients with congestive heart failure. In addition to inotropic effects, it seems to have immunosuppressive action. We tested the hypothesis that VES suppresses graft rejection, inotropic dysfunction caused by early rejection, and chronic coronary obstruction in a heterotopic rat cardiac transplantation model. METHODS: (1) To study acute rejection, hearts from Lewis-Brown Norway (LBN) rats were transplanted into Lewis rats, which were treated with or without VES (50 or 100 mg/kg/day orally). (2) In a functional study, LBN hearts with or without VES (100 mg/kg/ day) were isolated and perfused on day 3 after transplantation to assess inotropic response to isoproterenol (3 x 10(-8) M). (3) To study chronic rejection, Lewis hearts were transplanted into Fisher 344 rats, which were treated with or without VES (50 mg/kg/day) for 90 days. Coronary obstructive disease was assessed by morphometric analysis. There were five to six animals in each group. RESULTS: (1) VES (100 mg/kg/day) prolonged LBN heart survival (11.7 +/- 0.7 vs. 9.6 +/- 0.7 days in control; P < 0.05). (2) Left ventricular developed pressure was depressed in transplanted hearts regardless of VES treatment (84 +/- 12, 90 +/- 8 vs. 144 +/- 16 mmHg in untransplanted hearts; P < 0.01). The developed pressure after administration of isoproterenol in VES-treated hearts (184 +/- 20 mmHg) was higher than transplanted hearts without VES (118 +/- 16 mmHg; P < 0.05), and similar to untransplanted hearts (203 +/- 27 mmHg; P = NS). (3) Transplanted hearts treated with or without VES showed similar grades of rejection (2.0 +/- 0.3 vs. 2.6 +/- 0.2; P = NS), intimal area (6,996 +/- 3,186 vs. 13,441 +/- 5,165 microns2; NS), and coronary luminal obstruction (45 +/- 16% vs. 67 +/- 14%; NS). CONCLUSIONS: VES produces mild prolongation in survival of rat heart grafts, but has no significant effect on chronic graft atherosclerosis. VES preserves the positive inotropic effects of isoproterenol that are otherwise deteriorated by early acute rejection.     

Influence of adjuvants on the induction of autoantibodies to the thyrotropin receptor

To determine the influence of adjuvant on the induction of antibodies to thyrotropin receptor (TSHR), we immunized BALB/c mice with a extracellular domain of the TSHR (ETSHR) protein in complete Freund's adjuvant (CFA), Titer Max (TM) and Gerbu. Similarly, control groups of mice were immunized with bovine serum albumin (BSA) in each of the different adjuvants. As determined by ELISA, ETSHR given along with CFA elicited high titers of antibodies to ETSHR which were mainly restricted to the IgG1 subclass. Mice immunized with ETSHR in TM also developed high titers of anti-ETSHR antibodies but had higher levels of both IgG1 and IgG2a. However, immunization with ETSHR in Gerbu resulted in low titers of antibodies, restricted to IgG1 subclass. Immunization of mice with BSA in each of the three adjuvants induced higher antibody titers to BSA. The subclass of antibodies in mice immunized with BSA in CFA and TM were predominantly IgG1 and IgG2a with lower levels of IgG2b, whereas in Gerbu treated group, antibody to BSA was restricted to IgG1 subclass. Analysis of specificity of antibodies against ETSHR, in mice immunized with ETSHR, revealed that irrespective of the adjuvant used, the dominant reactivity was against peptide 1 (AA 22-41) with weaker reactivity against several other. peptides. The only exception was in mice immunized with ETSHR in TM which also showed significant reactivity against peptide 23 (AA 352-371). Mice immunized with the ETSHR in CFA or in TM showed elevated levels of serum TSH binding inhibitory immunoglobulins (TBII). However, mice immunized with ETSHR in Gerbu, which had lower titers of antibodies to ETSHR, showed normal TBII levels. These studies showed that adjuvant composition could influence the titer, subclass and fine specificity of antibodies to ETSHR which in turn could affect the development of TBII activity.