End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that abut 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.     

The anabolic effects of parathyroid hormone on cortical bone mass, dimensions and strength--assessed in a sexually mature, ovariectomized rat model

We examined the effect of the pineal neurohormone melatonin (MLT) on protection from viral encephalitis. The antiviral activity of MLT was evaluated in normal mice inoculated with Semliki Forest virus (SFV) and in stressed mice injected with the attenuated non-invasive West Nile virus (WN-25). Administration of MLT (s.c.) daily from 3 days before through 10 days after virus inoculation reduced viremia and significantly postponed the onset of disease and death by 7 to 10 days. Moreover, MLT injection reduced mortality of SFV (10 PFU) inoculated mice from 100% to 44%. In mice inoculated with high dose of SFV (100 PFU), MLT postponed death and reduced mortality by 20%. In all of the surviving mice anti-SFV antibodies were detected 22 days after virus inoculation. Infection of mice stressed by either isolation or dexamethasone injection with WN-25 induced mortality of 75% and 50% respectively, which was reduced by MLT administration to 31% and 25%, respectively. The efficiency of MLT in protecting from lethal viral infections warrants further investigations on its mechanisms of action.     

Alterations of pH in response to increased dietary protein in cattle are unique to the uterus

This study was undertaken with two objectives: 1) to determine whether the effect of excess dietary protein on intrauterine pH in cattle is specific to the uterus or manifested in other bodily fluids and 2) to determine whether the effect of excess ruminally degradable protein on uterine pH can be ameliorated by substitution with a less-degradable protein source. Thirty-six Holstein cows in early lactation were fed isoenergetic total mixed rations that either 1) met undegradable intake protein (UIP) and degradable intake protein (DIP) requirements (Balanced), 2) met DIP requirements and exceeded UIP requirements by 25% (High UIP), or 3) met UIP requirements and exceeded DIP requirements by 25% (High DIP). After diets had been fed > or = 2 wk, uterine, blood, salivary, and urinary pH and plasma urea nitrogen were determined at estrus (d 0) and d 7. Plasma urea nitrogen (mg/dL) was not different between estrus and d 7 but was significantly affected by diet (Balanced, 16.1 +/- 2.3; High UIP, 19.2 +/- 1.6; High DIP, 22.3 +/- 2.6; P < .05). There was no effect of treatment on the pH of any fluid measured at estrus: intrauterine, blood, salivary, and urinary pH averaged 6.84 +/- .05, 7.39 +/- .01, 8.30 +/- .05, and 8.15 +/- .05, respectively. In contrast, on d 7, uterine pH was significantly lower in both high-protein groups, regardless of protein degradability (Balanced, 7.13 +/- .05; UIP, 6.95 +/- .04; DIP, 6.85 +/- .05; P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the efficacy of sequential intravenous-oral administration of pefloxacin in community-acquired lower respiratory tract infections in patients with underlying conditions

We studied the efficacy of sequential intravenous-oral pefloxacin therapy in community-acquired lower respiratory tract infection in 24 patients with one or more underlying conditions. Twenty-eight patients were enrolled into the study but only 24 patients were evaluated. There were 16 males and 8 females with a mean age of 66.9 +/- 11.2 years (mean +/- SD, range 46 to 87 years). The underlying conditions present were bronchiectasis, chronic obstructive lung disease and diabetes mellitus. Patients who were older than 70 years but without any underlying condition were also enrolled. All received 4 days of intravenous pefloxacin 400 mg twice a day followed by oral pefloxacin 400 mg twice a day for another 10 days. Assessment of success was based on clinical, microbiological and radiological improvement. Pefloxacin produced 79.2% clinical cure rate. Another 8.3% showed improvement. Pefloxacin was well tolerated. There were few adverse effects and none of the patients required a change of antibiotic. Pefloxacin was an effective and well tolerated treatment for respiratory tract infection and had the advantage of broad in-vitro antibacterial activity, twice daily dosing and sequential availability in an intravenous and oral formulation.     

Isolation of a pentraxin-like protein from rainbow trout serum

To investigate the outcome of Graves' thyrotoxicosis after antithyroid drug management, data from 81 patients, treated in Chang Gung Memorial Hospital at Taipei and Linkou from October 1981 to March 1990, were analyzed. The gender ratio of female to male was 59:22. The mean age of onset was 33.1 +/- 10.5(15-60) year-old. All the patients were treated with antithyroid drug (Thionamide group) for a duration of 11 to 63 months (mean +/- SD = 28.1 +/- 9.8 months). Forty of 81 patients (49.4%) were remained remission after up to 2 years of follow-up. Those patients relapse usually occurred within 2 years after discontinuation of treatment (34/41), and only one exceptional case relapsed after 3 years. Three conditions affected the relapse rate. Patients with larger goiter (grade II-III) and shorter duration of treatment (< 23 months) had a higher relapse rate than those-with smaller goiter (grade O-I) [29/46 vs. 12/35; chi 2 = 6.576, p = 0.010; p = 0.015 in stepwise logistic regression (LR)] and longer duration of treatment (> or = 23 months) (15/20 vs. 26/61; chi 2 = 6.316, p = 0.012; p = 0.020 in LR). Patients with higher pre-treated serum triiodothyronine (T3) level (T3 > or = 300 ng/dl) had a higher relapse rate than those with lower T3 level (T3 < 300 ng/dl) in univariate analysis (30/50 vs. 11/31, chi 2 = 4.601, p = 0.032), but no significant difference by LR (P = 0.094). Other clinical parameters including age, sex, past history, family history, thyroxine (T4) level, T3/T4 ratio, thyroid autoantibodies, staging of ophthalmopathy, responsiveness to thyrotropin-releasing hormone stimulation test at the end of treatment, and whether combined treatment with thyroxine had no significant difference between the relapse and remission groups. These data suggest: (a) patients with larger goiter (grade II-III had higher relapse rate; (b) most of the recurrent thyrotoxicosis patients relapsed within two years after drug withdrawal; (c) continuing treatment for more than twenty-three months produces better outcome; (d) patients with Graves' thyrotoxicosis should be followed up for at least three years after withdrawal of antithyroid drug.     

Hyperdynamic circulation of cirrhotic rats: role of substance P and its relationship to nitric oxide

BACKGROUND: It has been suggested that excessive formation of nitric oxide (NO) is responsible for the hyperdynamic circulation observed in portal hypertension. Substance P is a neuropeptide partly cleared by the liver and causes vasodilatation through the activation of the endothelial NO pathway. However, there are no previously published data concerning the plasma level of substance P in cirrhotic rats and its relationship to NO. METHODS: Plasma concentrations of substance P and nitrate/nitrite (an index of NO production) were determined in control rats and cirrhotic rats with or without ascites using an enzyme-linked immununosorbent assay and a colorimetric assay, respectively. In addition, systemic and portal hemodynamics were evaluated by a thermodilution technique and catheterization. RESULTS: Cirrhotic rats with and without ascites had a lower systemic vascular resistance (2.6 +/- 0.2 and 3.9 +/- 0.4 mmHg ml(-1) x min x 100 g body weight, respectively) and higher portal pressure (14.6 +/- 0.6 and 11.3 +/- 1.8 mmHg) than control rats (6.5 +/- 0.3 mmHg x ml(-1) x min x 100 g BW and 6.8 +/- 0.2 mmHg, respectively, P < 0.05), and cirrhotic rats with ascites had the lowest systemic vascular resistance. Plasma levels of nitrate/nitrite progressively increased in relation to the severity of liver dysfunction (control rats, 2.7 +/- 0.5 nmol/ml; cirrhotic rats without ascites, 5.6 +/- 1.3 nmol/ml; cirrhotic rats with ascites, 8.3 +/- 2.2 nmol/ml; P < 0.05). Cirrhotic rats with ascites displayed higher plasma values of substance P (57.7 +/- 5.9 pg/ml) than cirrhotic rats without ascites (37.9 +/- 3.1 pg/ml, P < 0.05) and control rats (30.1 +/- 1.0 pg/ml, P < 0.05). There was no significant difference in plasma substance P values between control rats and cirrhotic rats without ascites (P > 0.05). No correlation was found between plasma levels of substance P and nitrate/nitrite (r = 0.318, P > 0.05). CONCLUSIONS: Excessive formation of NO may be responsible, at least partly, for the hemodynamic derangements in cirrhosis. Although substance P may not participate in the initiation of a hyperdynamic circulation in cirrhosis, it may contribute to the maintenance of the hyperdynamic circulation observed in cirrhotic rats with ascites.