Saliva collection by using filter paper for measuring cortisol levels in dogs.

Four experiments were conducted to evaluate the accuracy and reliability of noninvasive evaluation of cortisol in saliva of dogs. In experiment 1, we measured the cortisol concentration in the filter paper on which 250-µL cortisol solutions had been quantitatively pipetted and in filter papers dipped in cortisol solution. In experiment 2, we collected the blood and saliva of dogs 3 times at 30-min intervals and compared the cortisol concentrations to examine whether the dynamics of cortisol in the blood and saliva are similar. The results of experiments 1 and 2 showed that the cortisol concentration can be quantitatively measured with this method and that the dynamics of cortisol concentration in the plasma and saliva collected by using filter paper are not different (P = 0.14 for experiment 1 and P = 0.51 for experiment 2). In experiment 3, to investigate the factors related to inducing stress in dogs by using the filter-paper method of collecting saliva, we compared the cortisol concentrations at 0 and 30 min after collecting the saliva of pet dogs. The dog owners completed a survey on their dogs, providing basic information and reporting the collection of their dog's saliva. We found that the cortisol concentrations increased significantly in dogs whose owners spent >2 min collecting saliva (P = 0.005), suggesting that prompt collection of saliva is necessary for accurate assessment of cortisol without induction of a stress response. In addition, the cortisol concentrations increased significantly in dogs whose teeth were not regularly brushed (P = 0.04), suggesting that regular teeth brushing mitigates the effect of the collection process on cortisol concentrations in the saliva, with minimal stress to the dogs. In experiment 4, we measured cortisol concentrations in pet dogs accustomed to having their teeth brushed by their owners, before and after interaction with their owners, to assess whether brushing induces stress in dogs. We detected that the cortisol concentrations significantly decreased after human-dog interaction (P = 0.008), suggesting that this method does not induce stress in dogs. Our study indicates that the method of saliva collection by using filter paper is effective in measuring the cortisol concentrations to evaluate stress, although certain steps are required to enhance accuracy.

Furan ring oxidation strategy for the synthesis of macrosphelides A and B.

By using the convenient protocol for conversion of 2-substituted furans into 4-oxo-2-alkenoic acids ((i) NBS, (ii) NaClO(2)), macrosphelide B (2) was synthesized from furyl alcohol 5 (>98% ee) and acid 6 (99% ee). The protocol was first applied to the PMB ether of 5 to afford acid 13b. On the other hand, DCC condensation of acid 6 with 5 gave 16 after deprotection of the TBS group. Condensation was again carried out between 13b and 16 to furnish the key ketone 17, which upon reduction with Zn(BH(4))(2) afforded anti alcohol 18 stereoselectively (15:1). After protection/deprotection steps, the furan 18 was converted to seco acid 3 by using the furan oxidation protocol mentioned above, and lactonization of 3 with Cl(3)C(6)H(2)COCl, Et(3)N, and DMAP afforded 22 (MOM ether of 2), which upon deprotection with TFA produced 2. Transformation of 22 to macrosphelide A (1) was then investigated. Although the chelation-controlled reduction of 22 should afford the desired anti alcohol 24, Zn(BH(4))(2) at <-90 degrees C gave a 2 approximately 1:1 mixture of anti/syn alcohols. On the contrary, reduction with NaBH(4) in MeOH at -15 degrees C produced the syn isomer 23 with >10:1 diastereoselectivity. Mitsunobu inversion of the resulting C(14)-hydroxyl group and deprotection of the MOM group with TFA afforded 1. Similarly, reduction of 2 with NaBH(4) afforded the C(14)-epimer of 1 stereoselectively. The observed stereoselectivity in the reductions of 22 and 2 could be explained on the basis of computer-assisted calculation, which showed presence of the low-energy conformers responsible for the stereoselective reduction. In addition, conversion of 2 to 1 was established, for the first time.

Voluntary nystagmus associated with accommodation spasms.

BACKGROUND: Voluntary nystagmus has been recognized as a pendular, rapid, conjugate, primarily horizontal, benign eye movement initiated and maintained by voluntary effort. CASE: A 10-year-old Japanese girl presented with voluntary nystagmus associated with accommodation spasms. Her chief complaints, intermittent blurred vision, headache, and soreness of the eyes, were thought to be related to the voluntary nystagmus and accommodation spasms. FINDINGS: The waveform of the nystagmus appeared pendular, the frequency was 13-15 Hz, and the amplitude was 3-5 degrees. Scanning laser ophthalmoscopic video images clearly demonstrated vertical and torsional components in addition to the horizontal eye movements. Her refraction was unstable, varying between -0.5 diopters (D) and -5.5 D, and the recording of the accommodometer increased to -12.0 D when nystagmus was initiated. CONCLUSIONS: This may be a unique form of voluntary nystagmus that consists of horizontal, vertical, and rotational components associated with accommodation spasms. Observation of this patient continues, without any further treatment or examination.

Expression of Fos protein in the limbic regions of the rat following haloperidol decanoate.

To identify sites of antipsychotic drug action, the effects of acute and chronic haloperidol treatment on Fos protein expression in rat brain regions were examined by immunohistochemical methods. Male Wistar rats were injected with haloperidol decanoate (40 mg/kg, i.m. ) or vehicle. Fourteen days after injection, each rat was given an acute subcutaneous injection of haloperidol (0.25 mg/kg) or vehicle, and was transcardially perfused 2 h after the second injection. A single dose of haloperidol to chronic vehicle-treated rats produced significant increases in Fos-positive neurons in 18 of 21 brain regions examined including the several cortical areas, caudate-putamen, nucleus accumbens, lateral septum, thalamic nuclei, amygdala, hippocampus CA1, mesencephalic dopaminergic nuclei, and periaqueductal grey. The rats treated with acute vehicle after chronic haloperidol showed persistent Fos increases in confined brain regions comprising the lateral and central amygdala, lateral septum, and entorhinal cortex. Additional haloperidol injection to the chronic haloperidol-treated rats induced significant increases in Fos immunoreactivity in more widespread limbic-thalamo-cortical areas, whereas no significant increase was seen in the dorsolateral caudate-putamen. The persisting effects of haloperidol in the limbic and related structures, especially the amygdala, lateral septum, and entorhinal area may be of significance to the efficacy of long-term haloperidol treatment.

Effect of a titanium surface on bone marrow-derived osteoblastic cells in vitro.

The initial interaction between a titanium implant and the surrounding cancellous bone tissue was investigated by means of in vitro cultures of bone marrow-derived osteoblastic clone TMS-12 cells. Proliferation of TMS-12 cells cultured on a titanium surface was significantly reduced compared with that of control cells cultured directly on plastic, but alkaline phosphatase activity was the same as control cells. Co-culture of spleen cells with TMS-12 cells in the presence of a 1 alpha,25-dihydroxyvitamin D3 on titanium resulted in significant inhibition of tartrate-resistant acid phosphatase activity compared to control cultures on plastic. More prostaglandin E2 (PGE2) was produced by cells grown on a titanium surface than on plastic tissue-culture plates. On the other hand, mouse calvaria-derived osteogenic MC3T3-E1 cells, known to proliferate well on titanium, released similar amounts of PGE2 on titanium surfaces as on plastic tissue culture plates, indicating additional marked differences between the two osteoblastic cell types in response to a titanium surface. These results suggest that the reaction of bone marrow-derived osteoblastic cells to a titanium surface may fundamentally differ from that of calvaria-derived osteoblastics even though both cells are from bone tissue.

A new murine lymphocytotoxic monoclonal antibody recognizing HLA-A2, -A28 and -A9.

Monoclonal antibodies recognizing polymorphic as well as monomorphic epitopes on HLA antigens are important tools for understanding the immunobiology of HLA molecules. We immunized BALB/c mice with a HLA-A2 transfectant and screened for hybridomas which reacted with a HLA-A2 transfectant but not with a HLA-B75 transfectant. After subcloning by limiting dilution four times, a hybridoma secreting a monoclonal antibody (mAb) (IgG 2a, kappa) designated 1-145 was established. 1-145 reacted with Epstein-Barr virus transformed B lymphoblastoid cell lines (B cell lines) which expressed HLA-A2, -A28, -A23 and -A24. The titer of 1-145 in culture supernatant against HLA-A2 and -A28 antigens was similar and the titer against HLA-A23 was lower. 1-145 reacted with cells expressing HLA-A24 but the titer against HLA-A24 antigens was even lower than that against HLA-A23 antigens. The HLA-A24 antigens on the peripheral blood lymphocytes were not detected by 1-145 possibly due to the lower expression compared to the B cell lines. These differences of the titers were reflected to microlymphocytotoxicity assay in which 1-145 culture supernatant lysed all PBLs expressing HLA-A2,-A28 and -A23 but did not lyse PBLs expressing HLA-A24. Published deduced amino acid sequence data of HLA class 1 molecules indicate that Lys in position 127 may be critical for 1-145 binding.

Hypofrontality does not occur with 6-hydroxydopamine lesions of the medial prefrontal cortex in rat brain.

This study examined the effect of lesions of dopamine (DA) nerve terminals the medial prefrontal cortex on local cerebral glucose utilization (LCGU) and dopamine metabolism in the rat brain. Bilateral 6-hydroxydopamine lesions were stereotaxically placed in the medial prefrontal cortex. Twenty-eight days after the lesion, concentrations of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were determined in eight brain regions with a high-performance liquid chromatographic assay. LCGU was assessed by [14C]2-deoxy-D-glucose autoradiography. The lesion produced a striking reduction in DA (to 6% of the control value), and a moderate reduction in DOPAC and HVA in the medial prefrontal cortex. The ratio of DOPAC to DA in the medial prefrontal cortex was significantly elevated in the 6-OHDA lesioned animals. In contrast to DA depletion, LCGU in the medial prefrontal cortex of the lesioned rats was unaltered when compared with the control. These findings suggest that decreased energy metabolism in the frontal cortex, i.e., hypofrontality, does not occur with decreased DA innervation of that site.

Comparative study of dopamine metabolism with local cerebral glucose utilization in rat brain following the administration of haloperidol decanoate.

The effects of haloperidol decanoate on dopamine (DA) metabolism in discrete regions of rat brain were investigated and compared with changes in local cerebral glucose utilization (LCGU). The concentration of DA and its metabolite, homovanillic acid, and the alpha-methyl-p-tyrosine (alpha-MT)-induced decline of DA were measured in 6 brain regions by a high-performance liquid chromatographic assay. LCGU in 26 brain regions were examined by [14C]2-deoxy-D-glucose autoradiography. At 24-hr after intramuscular injection of haloperidol decanoate (60 mg eq/kg to haloperidol), the concentration of homovanillic acid in the prefrontal cortex, caudate-putamen, accumbens nucleus, lateral amygdala, and medial thalamus showed significant increase compared with control values. On day 21, the increase in these regions was significantly attenuated with no significant difference from the controls. Furthermore, chronic haloperidol rats showed alpha-MT-induced decline of DA to a similar extent in the control rats. LCGU on day 21 showed significant decrease in the parietal cortex, and a tendency toward decrease in the prefrontal cortex, lateral amygdala and medial thalamus compared with the controls. There was no significant change in LCGU in the caudate-putamen or accumbens nucleus. Chronic haloperidol would thus appear to affect energy metabolism mainly in the cortico-thalamo-limbic circuits, and this may not correspond well to presynaptic DA metabolism.

Expression on outer membranes of mannose residues, which are involved in osteoclast formation via cellular fusion events.

Osteoclast, the bone-resorbing cell, is formed from hematopoietic precursors via cell-cell fusion. To evaluate the possibility that under certain specific conditions mannose residues may be expressed on the mammalian cell surface, we examined the action of pradimicin derivatives, which bind specific sugars such as the mannose residue, on the formation of osteoclast induced in the coculture of mouse spleen cells with mouse stromal cells, a process in which cell-cell fusion is involved. Osteoclast formation was inhibited by treatment of this coculture system with pradimicin at the later stage (day 4-7), and this inhibition was specifically abrogated by mannose-rich yeast mannan. During the 8-day cocultivation, osteoclast formation was blocked by the pradimicin on days 6 and 7, when mononuclear preosteoclasts fused into multinucleated osteoclasts. With an interactive laser cytometer ACAS570, fluorescein isothiocyanate-labeled pradimicin was observed to bind osteoclast progenitors at the fusion stage and to have no binding affinity for osteoclast progenitors at the early stage (day 0-3) or for osteoclasts, which were formed after performing fusion between mononuclear preosteoclasts. These results suggest that mannose residues were expressed on outer membranes of monocytes under pathophysiological conditions and that they were involved in the osteoclast formation via cellular membrane fusion events.

Involvement of adhesion molecules LFA-1 and ICAM-1 in osteoclast development.

We report here that leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) are involved in osteoclast development. Osteoclast development was observed on co-culture of mouse spleen cells and mouse bone marrow derived clonal stromal cells, TMS-14, in the presence of 1 alpha, 25-dihydroxyvitamin D-3 (1 alpha, 25-(OH)2D3) for 8 days, and quantified with respect to tartrate-resistant acid phosphatase (TRACP) activity. When either one of the monoclonal antibodies (MAbs) to mouse LFA-1 and mouse ICAM-1 was added to the co-culture system, the TRACP activity was significantly inhibited. The experiment in which one-day treatment with each of these MAbs was performed during the 8 days of cultivation showed that the inhibitory effects of both MAbs on the TRACP activity at 8 days were observed from an early stage of the culture, but were more notable at a later stage (days 4-6). As the expression of ICAM-1 was observed on both spleen cells and TMS-14, we next examined whether the interaction between stromal cells and osteoclast progenitors or among osteoclast progenitors was more important for osteoclast development. To determine this, rat spleen cells and a MAb to rat ICAM-1 were used instead of those of mouse. When MAb to rat ICAM-1 or mouse ICAM-1 was added to the co-culture system of rat spleen cells and TMS-14, the inhibitory effect of the MAb to rat ICAM-1 was mainly observed at a later stage of the culture period and that of anti-mouse ICAM-1 antibody was only observed at an earlier stage. These results indicate that adhesion molecules LFA-1 and ICAM-1 may play a role in osteoclast development via interaction between stromal cells and osteoclast progenitors as well as among osteoclast progenitors.

A human monoclonal antibody that detects HLA-A1, A23 and A24 antigens.

We report the production and characterization of a human monoclonal IgM (mu, kappa) antibody recognizing the HLA A1, A23 and A24 antigens. B lymphocytes obtained from a multiparous Japanese woman were transformed in vitro by Epstein-Barr virus, screened with an immune adherence assay, and fused with a murine myeloma cell line, P3-X63-Ag8.653. After subcloning by limiting dilution three times, a stable antibody-secreting hybridoma cell line, 4-35-7, was identified. The culture supernant had a titer of 1:32-64 against each of A1-, A23- and A24-positive lymphocyte panels, and showed complete correlation (r = 1.00) with the A1, A23 and A24 antigens on a lymphocyte panel of 287 unrelated, class I HLA-typed donors by the NIH cytotoxicity assay. Monoclonality of the antibody was ensured by Southern blot analysis of the human immunoglobulin heavy chain gene of 4-35-7. In view of the published data on HLA class I nucleotide sequences, the antibody may recognize an antigeneic determinant including two amino acid residues, Asp-166 and Gly-167, in the alpha 2 helix of the class I molecule that are specific for A1, A23 and A24 so far analyzed.

Ipriflavone inhibits murine osteoclast formation in vitro.

Ipriflavone, one of the isoflavone derivatives, is a therapeutic drug for osteoporosis. The mechanism is thought to be the inhibition of bone resorption. In the present paper, we report that ipriflavone inhibited formation of osteoclasts from murine spleen cells co-cultured with stromal cells cloned from murine bone marrow. In this system, ipriflavone inhibited osteoclast generation in a dose-dependent manner (10(-7)-10(-5) M). Ipriflavone also inhibited prostaglandin E2 production in MC3T3-E1 cells, which are widely employed as osteoblasts. Moreover, ipriflavone inhibited the proliferation of stromal cells (10(-6)-10(-5) M), but not osteoblastic cells. These results suggest that one mechanism for the inhibitory effects of ipriflavone on bone resorption is the inhibition of osteoclast formation through inhibiting prostaglandin E2 production in osteoblasts and thereby suppressing proliferation of stromal cells.