Walker 256/S carcinosarcoma causes osteoporosis-like changes through ectopical secretion of luteinizing hormone-releasing hormone.

We have shown that Walker 256/S mammary carcinoma caused osteoporosis-like changes in young female rats, accompanied by low serum estradiol and hypercalciuria without changes in the serum levels of calcium, phosphorus, and parathyroid hormone-related peptide. In this study, we investigated the cause of bone loss after Walker 256/S inoculation into female 6-week-old Wistar Imamichi rats, focusing on the sex hormone balance in the host animal. Walker 256/S-bearing rats showed characteristic osteoporosis, with a significant increase in spleen weight and a significant decrease in uterine weight by 14 days after s.c. tumor inoculation. In the in vitro bone marrow culture, mineralized nodule formation ability decreased according to the time after tumor inoculation, and tartrate-resistant acid phosphatase-positive multinucleated cell formation increased at 7 days after tumor inoculation, but it began to decrease at 14 days after tumor inoculation. This indicates that after inoculation with Walker 256/S tumor, the progenitors of osteoblasts and ostroclasts lost their balance in the bone turnover, resulting in bone resorption. On the other hand, Walker 256/S carcinoma expressed luteinizing hormone-releasing hormone (LH-RH) mRNA, and in Walker 256/S-bearing rats, the serum LH-RH level increased significantly from 3 days after tumor inoculation, whereas in the healthy control rats, this level was very low. Consequently, the serum levels of follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone were significantly lower in the tumor-bearing rats than in the healthy control rats. Because the LH-RH gene is located in the long prolactin release-inhibiting factor (PIF) gene and mRNA amplified by reverse transcription-PCR in this study contained whole LH-RH and a part of PIF, the Walker 256/S tumor is thought to express PIF. Indeed, the serum prolactin level decreased in tumor-bearing rats. The serum level of growth hormone, one of the other pituitary hormones, was not changed. Moreover, the level of an osteolytic cytokine, tumor necrosis factor alpha, increased in the serum of Walker 256/S-bearing rats, although this may be a result of the immune response of the host animal to tumor growth as well as an enlarged spleen. In conclusion, the Walker 256/S tumor lowers estrogen secretion through ectopical oversecretion of LH-RH, and then osteolytic cytokines, such as tumor necrosis factor alpha, increase in tumor-bearing rats, escape the control of estrogen, and activate osteoclasts, resulting in bone loss in a short period.

Selective drug delivery system to bone: small peptide (Asp)6 conjugation.

Targeting a drug on hydroxyapatite (HA) could be a promising way for selective drug delivery to bone, because HA, an inorganic component in hard tissues (bone and teeth), does not exist in soft tissues. Several bone noncollagenous proteins, which bind to HA, have repeating sequences of acidic amino acids in their structures as possible HA-binding sites. Thus, we think that a small peptide of repetitive acidic amino acid could work as a carrier for selective drug delivery to the bone. To test this hypothesis, we conjugated (Asp)6 to fluorescein isothiocyanate (FITC), evaluated its affinity to HA in vitro, and examined its tissue distribution after injection into rats. Although fluorescein itself did not bind to HA, (Asp)6-FITC bound to HA as well as calceine and tetracycline. Twenty-four hours after intravenous injection of (Asp)6-FITC to rats, animals were killed, and ground sections of hard tissues and cryosections of soft tissues were made. Under a confocal laser scanning microscope, clear labeling lines were observed in bones and teeth, whereas no labeling was detected in soft tissues. In the rats administered with fluorescein alone, the fluorescent labeling was detected in neither hard nor soft tissues. Fluorescent analysis of blood, urine, and bones after (Asp)6-FITC administration revealed that biological half-life of FITC in blood was short (60 minutes) and that within 24 h, 95% of the administered FITC was excreted as urine whereas 2% of the FITC accumulated in bones. After subcutaneous administration of (Asp)6-FITC to mice, fluorescent intensity remaining in the femurs was measured periodically. In these mice the biological half-life of FITC in the femur was 14 days. Present results indicate that (Asp)6 is effective as a carrier for selective drug delivery to bone.

Selective delivery of estradiol to bone by aspartic acid oligopeptide and its effects on ovariectomized mice.

We have developed a novel osteotropic prodrug of estradiol (E(2)) conjugated with L-Asp-hexapeptide (E(2).3D(6)), which has very low affinity for estrogen receptors, and in this study, we examined its pharmacokinetic behavior and pharmacological potential. After a single iv injection of E(2) x 3D(6) to mice, the half-time for elimination from plasma was about 100 min; however, E(2) was selectively delivered to the bone and eliminated very slowly, declining to the endogenous level at about 7 days. After a single iv injection of E(2), the half-time in plasma was about 70 min, whereas E(2) was highly distributed to the uterus, and the bone concentration of E(2) was only slightly increased at 6 h. When E(2) (0.37 micromol/kg, sc, every third day) or E(2) x 3D(6) (0.11 to 1.1 micromol/kg, sc, every seventh day) was administered to OVX mice for 4 weeks, E(2) increased the bone mineral density (BMD) together with weights of liver and uterus, whereas E(2) x 3D(6) increased only the BMD, in a dose-dependent manner. E(2) x 3D(6) enhanced the expression of messenger RNAs of bone matrix proteins (osteopontin, bone sialoprotein, type I collagen alpha) of OVX mice at 4 h after administration, but E(2) did very slightly. These results indicate that the E(2) prodrug was delivered to the bone, where it gradually released E(2), thereby ameliorating bone loss. This acidic oligopeptide appears to be a good candidate for selective drug delivery to bone.

Selective inhibitors of cyclic AMP-specific phosphodiesterase: heterocycle-condensed purines.

To reverse the adverse reactions of alkylxanthines and to develop novel inhibitors of cyclic AMP-specific phosphodiesterase (PDE IV), a series of heterocycle-condensed purines were designed and synthesized. Some of these new compounds had similar or more potent and selective inhibitory activity against PDE IV than known PDE IV inhibitors. The tracheal-relaxant activity of these compounds was closely correlated with their PDE IV-inhibitory activity. Moreover, these purine analogues did not have any positive-chronotropic action or adenosine-antagonistic action on isolated heart preparations, which are the particular adverse reactions of alkylxanthines. Among them, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (1c), which was the most selective and potent PDE IV inhibitor, did not cause emesis in Suncus murinus at a dosage range of 10-100 mg/kg (po), while an imidazole analogue of 1c (4c) and known PDE IV inhibitors such as rolipram and denbufylline caused emesis even at 10 or 30 mg/kg.

Reduction of bone loss by denbufylline, an inhibitor of phosphodiesterase 4.

The effects of denbufylline, a xanthine derivative with selective inhibitory activity on the phosphodiesterase (PDE) 4 isoenzyme, on bone loss in Walker 256/S-bearing rats and on mineralized nodule formation and osteoclastlike cell formation in bone marrow culture systems were examined. Serial oral administrations of denbufylline inhibited the decrease in the bone mineral density of femurs from Walker 256/S-bearing rats, without influence on the healthy rats. Denbufylline restored the bone mass and the number of osteoclasts and osteoblasts per trabecular surface in the femur metaphysis. Among PDE inhibitors, only PDE4-selective inhibitors increased the number of mineralized nodules and decreased the number of osteoclastlike cells in the in vitro bone marrow culture systems, and dibutyryl cyclic AMP mimicked these effects in the in vitro systems. These results suggest that the PDE4 isoenzyme may play an important role in bone turnover through cyclic AMP and that its inhibitors are candidates for therapeutic drugs for the bone loss diseases.

Regional lung hematocrit variation and assessment of acute lung injury.

Estimating blood content in the lung remains a key step in calculating lung water volume and microvascular permeability. We studied the effect of regional lung hematocrit (Hct) variation on assessment of acute lung injury. Escherichia coli endotoxin was administered in guinea pigs intravenously. Lung injury was evaluated by measuring the wet-to-dry weight ratio (W/D) and transvascular 125I-labeled albumin leakage for 3 h [tissue-to-plasma 125I-albumin ratio (T/P)] in five tissue samples from each animal. Residual blood content was corrected using either 51Cr-red blood cells as a blood cell marker, 99mTc-albumin as a plasma marker, or both, injected 10 min before the guinea pigs were killed. Lung Hct, estimated from the marker counts of lung and peripheral blood samples, was lower than peripheral blood Hct; intraindividual variation, represented by the standard deviation in each subject, was 0.024 +/- 0.015 for the control group (coefficient of variation 8.0 +/- 5.1%) and 0.026 +/- 0.013 for the endotoxin group (coefficient of variation 8.5 +/- 4.1%). Uncorrected W/D for residual blood content was greater than the corrected W/D. 99mTc-albumin correction gave values closer to the W/D corrected by both markers. T/P corrected by 99mTc-albumin showed smaller data variations than the values obtained with 51Cr-red blood cell correction, which was affected by variations in lung Hct. We recommend using a plasma marker to correct for blood content in assessing acute lung injury by W/D and T/P.

Attenuation of hyperoxic lung injury by the 21-aminosteroid U-74389G.

Hyperoxic lung injury is attributable to oxygen radicals produced under hyperoxic conditions. The 21-aminosteroid (AS), U-74389G, is a potent antioxidant. We examined the effect of U-74389G on lung injury in guinea pigs during exposure to 90% O2 for 48 h. We injected either vehicle or 10 mg/kg of U-74389G 30 min before the O2 exposure and injected the same dose 12, 24, and 36 h later. We performed two series of experiments after exposure. In the first series, we measured the clearance rate of 99mTc-labeled dialdehyde starch (DAS) from the lungs as an index of pulmonary epithelial damage in three experimental groups consisting of 1) control (n = 6) O2 alone (n = 6), and 3) O2 + AS (n = 6). In the second series, pulmonary endothelial injury was estimated by using 28 guinea pigs divided into four experimental groups consisting of 1) control (n = 8), 2) AS alone (n = 5), 3) O2 alone (n = 6), and 4) O2 + AS (n = 9). In the second series, we measured the wet-to-dry weight ratio (W/D) as an index of lung water and the concentration ratio of 125I-labeled albumin in lung tissue and bronchoalveolar lavage (BAL) fluid compared with plasma (T/P and BAL/P, respectively) as indexes of pulmonary endothelial damage. Cell accumulation in BAL fluid and lung tissue samples was also assessed in the second series.(ABSTRACT TRUNCATED AT 250 WORDS)

The neuromodulatory effects of VIP/PACAP on PC-12 cells are associated with their N-terminal structures.

ONOUE, S., WAKI, Y., NAGANO, Y., SATOH, S., KASHIMOTO, K. Neuromodulatory Effects of VIP/PACAP on PC-12 Cells Are Associated with Their N-terminal Structures. PEPTIDES xx(xx) 000-000, 200x.- The current study explored whether the differences in biological activities in PC-12 cells between vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are attributable to the sequence difference in their N-terminal portions and are correlated with the solution structures of the peptides. In the neurite outgrowth assay, N-terminal modification of VIP to PACAP-like sequences altered its effect, the activity was confirmed even at a low concentration (10(-10) M). On the contrary, N-terminal modification of PACAP 27 to VIP-like sequences reduced its activity. These relationships were also confirmed for the inhibitory effects of the peptide analogues on PC-12 cells growth at 10(-7) M. The present results combined with our previously reported data, including binding assay, support that the N-termini of VIP/PACAP plays an important role in their activities.

Glucose in the dialyzate does not reduce the free amino acid loss during routine hemodialysis of non-fasting patients.

In order to clarify the effect of dialyzate glucose on free amino acid loss during routine 5 hr hemodialysis (HD), 10 stable non-fasting HD patients were dialyzed twice using dialyzates (Na 141, K 2.5, Ca 3.5, Cl 101, Mg 1.5, Acetate 36 mEql) with (500 mg/dl) or without glucose. Total free amino acid loss in the dialyzate and plasma free amino acids were measured using a liquid chromatograph amino acid analyzer. Total free amino acid losses into the dialyzate with and without glucose were 6598 +/- 482, and 6482 +/- 174 mg respectively. This is not statistically significant. The plasma concentration of free amino acid fell considerably during dialysis, but there was no significant difference between the two types of dialyzates used. We concluded that the addition of glucose to the dialyzate does not reduce the free amino acid loss during routine HD of non-fasting patients. This result does not accord with a previous report that adding glucose to the dialyzate decreases the loss of free and bound amino acid during dialysis.

Clinical significance of measuring myeloperoxidase, thiobarbituric acid reactive material and 7S collagen in plasma of patients with adult respiratory distress syndrome.

We measured myeloperoxidase (MPO), thiobarbituric acid reactive material (TBARM), and Type IV collagen 7S domain (7S collagen) in the plasma of 21 patients with acute lung injury (ALI). Sixteen healthy subjects served as a control group. There was no significant difference in MPO between the control and ALI groups. The TBARM and 7S collagen concentrations in ALI (TBARM; 3.05 +/- 0.65 nMol/ml, 7S collagen 9.06 +/- 5.96 ng/ml: Mean +/- SD) were significantly higher than those in the control group (2.54 +/- 0.33 and 3.43 +/- 1.05, p < 0.05). TBARM and 7S collagen levels of deceased ALI patients were higher than those of surviving ALI patients (p < 0.05). There were significant correlations between the plasma levels of these two parameters and the lung injury scores. Our findings suggest that plasma TBARM and 7S collagen are useful markers for the assessment of the severity of ARDS.

Observation of the growth process of a beagle embryo and fetus by ultrasonography.

Detailed observations of the growth process of the embryos and fetuses (hereafter, referred to as just fetus) of 6 pregnant Beagle bitches (14-27 months old) was carried out using ultrasonography. From daily measurements of the transversal diameter of the fetal abdomen, diameter of the fetal head and diameter of the fetal heart, significant growth curves (secondary regression equations) were obtained from the day of gestation and the measured values (p < 0.01). The results of this study showed that this method can be applied to estimate the gestation age of dogs in cases when the mating day is unknown, and determine the state of fetal growth.

[Evaluation of ototoxic effect of micronomicin by intravenous drip infusion].

Ototoxic effect of micronomicin (MCR) in intravenous drip administration was investigated in guinea pigs (300--400 g) receiving MCR for 30 days at dose of 50 and 100 mg/kg, respectively. MCR was dissolved in physiologic saline and 1.5 ml of the solution was infused through polyethylene tube into the left external jugular vein with Perista mini-pump for 60 minutes every day after measurement of body weight. Auditory impairment was monitored by pinna reflex audiometry. Pinna reflex loss was not detected in any animal in frequency range (0.5 to 20 kHz). Cochlear hair cell damage generally was of mild degree. Two out of 6 animals treated with MCR 50 mg/kg (Table 1) showed outer hair cell loss confined to unilateral basal end and posterior 3/4 of the first turn, respectively. Outer hair cell loss was noticed in 4 of 9 animals receiving MCR 100 mg/kg (Table 2); much less extensive in 3 animals, unsymmetrical slightly extensive in remaining 1. Unilateral circumscribed loss of inner hair cells was noticed at lower part of the hook in the latter one. Vestibular hair cell loss was scattered and less extensive and occurred in 4 of the 6 50 mg/kg given animals and in 7 of the 9 100 mg/kg ones. Comparison in incidence and extension of the outer hair cell loss of the cochlea (Tables 3, 4) in the present study and previous ones on the ototoxic effect of MCR in intramuscular and intravenous administration suggests that there was no distinct difference in enhancement of the ototoxic effect in the intravenous drip administration.

[Clinicopathological studies on squamous cell carcinoma of the esophagus with esophageal gland-like transformation].

One hundred and seventy cancerous lesions observed among 159 esophagectomised specimens were systematically investigated with histochemical staining methods in addition to the ordinary hematoxylin-eosin staining. About 2/3 of them, 113 lesions, were entirely consisted of the squamous elements, regardless with pearl-formation or not, Group 1. On the contrary, the remaining 57 lesions (33.5%) showed foci of esophageal gland-like transformation, Group 2. Those lesions were specified by constituent epithelia with esophageal glandular epithelia-like characteristics; positive for epithelial mucin, secretory component and S-100 protein. Such esophageal gland-like structures could be demonstrated exclusively in cancerous foci penetrated through the lamina muscularis mucosa. Those 113 lesions of the Group 1 did not show any such transformation even by the meticulous examination of the whole lesion by the step sectioning. Those lesions of the Group 2 had a distinct tendency of the perpendicular mural penetration and submucosal extension than the horizontal mucosal involvement, giving rise a characteristic appearance resembling to submucosal tumors. On the contrary, those of the Group 1 showed an ordinary mural extension pattern corresponding to the extent of mucosal involvement, so-to-speak, a typical features of common esophageal cancer. No outstanding difference in biological behaviours could be obviated between two groups so far examined; survival rate, lymph node metastasis, vascular permeation, intraepithelial extension, etc.

Hypervitaminosis A: a contributing factor to anemia in regular dialysis patients.

In order to evaluate the toxicity of hypervitaminosis A in regular dialysis patients, the relationships between the plasma levels of vitamin A, retinol binding protein (RBP), prealbumin (PA), and biochemical parameters were studied in 47 patients. Plasma vitamin A levels were inversely correlated with hematocrit (r = -0.5), which was also significantly correlated with RBP/vitamin A ratio (r = 0.61). Immunoreactive parathyroid hormone (i-PTH) and very low density lipoprotein (VLDL) were inversely related to RBP/vitamin A and RBP/PA ratios. These findings suggest that a raised plasma vitamin A level in regular dialysis patients contributes to anemia and hypervitaminosis A toxicity.

Enhancement of the cytotoxic effect of mitomycin C by beta-adrenergic stimulants in rat ascites hepatoma AH130 cells.

Among several sympathomimetic drugs, isoproterenol, epinephrine and norepinephrine significantly potentiated the cytotoxic effect and the uptake of MMC into rat ascites hepatoma AH130 cells in in vitro experiments, but salbutamol, tulobuterol and phenylephrine did not. The enhancement of MMC uptake into AH130 cells by isoproterenol and norepinephrine was diminished by propranolol but not by phentolamine. These results indicate that the cytotoxic effect and the uptake of MMC is potentiated by the stimulation of beta-adrenoceptors, but not by stimulation of beta 2- and alpha-receptors.