Convergence of circadian and sleep regulatory mechanisms on hypocretin-1.

Hypocretin is a potential regulator of sleep and wakefulness and its levels fluctuate with the day-night cycle with high levels during the animal's activity period. Whether the daily fluctuations are driven endogenously or by external light cycles is unknown. We investigated the circadian and homeostatic regulation of hypocretin in the absence of environmental light cycles. To this purpose we performed repetitive samplings of cerebrospinal fluid in rats through implanted microcannulas in the cisterna magna and determined hypocretin-1 levels by radioimmunoassay. These experiments were also performed in rats that received a lesion of the suprachiasmatic nucleus (SCN), a major pacemaker for circadian rhythms in mammals. The results showed sustained rhythmicity of hypocretin in constant dim red light in control animals. SCN-lesioned animals showed no circadian rhythms in hypocretin and mean hypocretin levels were remarkably low. The results indicate that the SCN is indispensable for rhythmicity in hypocretin and induces a daily increase in hypocretin levels during the animal's active phase. Additional sleep deprivation experiments were carried out to investigate homeostatic regulation of hypocretin. Hypocretin levels increased in response to sleep deprivation in both control and SCN-lesioned animals, demonstrating that sleep homeostatic control of hypocretin occurs independently from the SCN. Our data indicate that the circadian pacemaker of the SCN and sleep homeostatic mechanisms converge on one single sleep regulatory substance.

The effect of exercise on systemic and bone concentrations of growth factors in rats.

Exercise can prevent bone loss and increase bone density. Growth factors such as insulin-like growth factor-I (IGF-I) and transforming growth factor beta (TGFbeta) are thought to be involved in the local response to mechanical loading, resulting in bone remodelling. We tested the effect of additional weight bearing during exercise on the systemic response of IGF-I and local bone response of IGF-I and TGFbeta. Thirty-four female Wistar rats (aged 3 months, weight 226.9 +/- 20.2 g) were randomly divided in four groups: group 1 baseline controls; group 2 sedentary controls; group 3 ran 15 min a day on a motor-driven exercise belt; group 4 ran 15 min a day with a backpack containing 40 g. The animals ran 5 days a week, for 6 weeks, with an average velocity of 16.6 +/- 4.4 m/min, and a slope of 5 degrees uphill. The serum growth hormone (GH) concentration was significantly higher in the running rats (group 3, P = 0.009) than in the sedentary controls (group 2). The IGF-I and IGF binding protein 3 (IGFBP3) levels in serum and the IGF-I levels in liver were similar in all groups. In the tibia no significant differences were observed in IGF-I, IGFBP3 and TGFbeta concentration. In the humerus, the IGF-I concentration was lower in the running rats (group 3) than in the sedentary controls (P = 0.04), but it was higher in the rats that ran with additional weight than in those without (P = 0.009). The TGFbeta concentration in the humerus was lower in both group 3 (P = 0.001) and 4 (P = 0.03) than in the sedentary controls. The effects in bone caused by mechanical stimulation cannot be explained by changes in serum IGF-I and IGF-I produced in the liver. The concentrations of IGF-I and TGFbeta in bone appeared to be modulated by running exercise.

Increase of decorin content in articular cartilage following running.

The effect of long distance running exercise (40 km/day for 15 weeks, five days a week) on the decorin content of articular cartilage from the knee joint was studied in female beagle dogs. Samples from load bearing sites on the lateral plateau of the tibia (TL), and pooled material from two minimum load bearing sites on the posterior section of lateral (FLP) and medial (FMP) femoral condyles were analyzed. The running exercise protocol did not lead to significant changes in the overall glycosaminoglycan content of the cartilage. However, the amount of decorin significantly increased in the TL samples, and also in the FMP pool. These results support earlier in vitro observations that decorin synthesis is stimulated by loading, independent of the synthesis of aggrecan.

Mechanical loading affects the synthesis of decorin and biglycan in intact immature articular cartilage in vitro.

Sesamoid bone cartilage from the metacarpophalangeal (MCP) joints of six-month-old calves was cultured intact on its bone support. The sesamoid bones for the experiment were subjected to loading with 0.2 MPa at 0.2 Hz for a week. Controls were cultured for 0 or 7 days, labelled with [35S] sulfate for 17 h, harvested, and extracted in 4 M guanidine HCI with proteinase inhibitors, for analysis on Sepharose CL-2B columns under dissociative conditions. It was found that the population of labelled small proteoglycans was significantly larger in the loaded cartilage than in the cultured controls. This population was pooled and further purified on dissociative Sepharose CL-4B columns. The resulting profiles showed two peaks. The material eluting at Kav 0.80 contained only chondroitin sulfate and keratan sulfate chains. The peak at Kav 0.48 was analysed by SDS-polyacrylamide gel electrophoresis. It consisted of the dermatan sulfate proteoglycans decorin and biglycan. The amounts of newly synthesized decorin and biglycan in the cultured control cartilage were lower than in the control cartilage that had been labelled on day 0. The synthesis of decorin, but not of biglycan, was significantly higher in the loaded cartilage than in the cultured control cartilage. Through its interaction with collagen type II, decorin may aid the adaptation of articular cartilage to increased loading.

The effects of loading on the synthesis of biglycan and decorin in intact mature articular cartilage in vitro.

Sesamoid bone cartilage from the metacarpophalangeal joints of 5-year-old cows was cultured intact on its bone support. The incorporation of sulfate increased similarly in experimental cartilage that was subjected to cyclic loading (0.2 MPa, 0.2 Hz) for a week and in control cartilage that was cultured without loading. The synthesis of a population of small macromolecules decreased in the cultured controls, but was maintained at a constant level in the experimental samples. This population was isolated through Sepharose CL-2B chromatography. Subsequent application to Sepharose CL-4B yielded two distinct peaks. One contained protein-free chondroitin sulfate, keratan sulfate, and possibly some dermatan sulfate glycosaminoglycans. The other, more prominent, peak consisted of dermatan sulfate proteoglycans. This peak material was pooled, and applied to a 4-15% SDS-gel. The material was separated into two major bands, which represented biglycan and decorin. They decreased to less than half their day-0 value, in the cultured control. In the loaded cartilage, biglycan synthesis remained constant while decorin synthesis increased. These findings suggest that biglycan and decorin are involved in the adaptation of articular cartilage to variations in loading regime.

A high-molecular-weight (greater than 8.10(5)) non-collagenous glycoprotein is synthesized by bovine cartilage in vitro.

A high-molecular-weight (greater than 8.10(5)) glycoprotein was detected in [3H]glucosamine-labeled bovine cartilage. Extraction with varying amounts of guanidinium chloride showed that the molecule was not tightly bound to other matrix substances. Enzyme digestions identified the molecule as a non-collagenous glycoprotein. This glycoprotein constituted 10-20% of the [3H]glucosamine-labeled macromolecular material that was released into culture medium on the first day after labeling. The 3H-labeled glycoprotein was purified by anion-exchange chromatography, CsCl gradient centrifugation and gel filtration. The purified glycoprotein appeared on an SDS-polyacrylamide gel as one slightly polydisperse band, which could not be reduced by beta-mercaptoethanol.

The stability of the nitrosated products of indole, indole-3-acetonitrile, indole-3-carbinol and 4-chloroindole.

The nitrosation rates of indole-3-acetonitrile, indole-3-carbinol, indole and 4-chloroindole and the stability of their nitrosated products were investigated. Each of the nitrosated indole compounds was directly mutagenic to Salmonella typhimurium TA100 in the following order of potency: 4-chloroindole much greater than indole-3-carbinol greater than or equal to indole greater than indole-3-acetonitrile. Total N-nitroso determinations, carried out according to a modified method of Walters et al. (Analyst, Lond. 1978, 103, 1127), and Ames test results revealed that each of the indole compounds immediately formed mutagenic N-nitroso products upon nitrite treatment under acidic conditions. However, the nitrosation rates of indole and 4-chloroindole were higher than those of indole-3-acetonitrile and indole-3-carbinol. For indole-3-carbinol, indole-3-acetonitrile and indole, no change in the amount of nitrosated products was observed at increasing incubation times from about 15 up to 60 min. For 4-chloroindole the amount of nitrosated products decreased with increasing incubation times. In all cases the responses in the Ames test paralleled the amounts of nitrosated products. The stabilities of the nitrosated products of the indole compounds were investigated at pH 2 and 8. Both mutagenicity data and measurements by high-performance liquid chromatography using a photohydrolysis detector indicated that the nitrosation products of indole-3-acetonitrile, indole-3-carbinol and indole were more stable at pH 8 than at pH 2. Conversely, nitrosated 4-chloroindole was stable at pH 2 but not at pH 8. The pH 8 chromatograms showed a large nitrite peak. From this we hypothesized that the presence of free nitrite might be responsible for the stability of nitrosated indole-3-acetonitrile, indole-3-carbinol and indole at pH 8. Experiments confirmed the existence of an equilibrium between the nitrosated indole compound and the free indole compound plus nitrite.

Consequences of failure of the reticular groove reflex in veal calves fed milk replacer.

A number of problems seen in veal calves are associated with insufficient adaptation to the changes in husbandry connected with the fattening system. These problems include chronic tympanitis, chronic vomiting, cachexia, unthriftiness, abomasal erosions and ulcers. Some of these problems are associated with failure of the reticular groove reflex, and result in ruminal drinking. Persistent ruminal drinking causes a syndrome characterised by a variety of symptoms such as unthriftiness, severe growth retardation, inappetence, recurrent tympany, abdominal distension, a long dry haircoat and clay-like faeces. Clinical studies performed to investigate the pathogenesis of the syndrome are described. It was found that the milk was stored in the rumen and was partly broken down. The presence of milk in the rumen induced typical changes in the ruminal mucosa (hyper/parakeratosis) and also inhibited casein clot formation in the abdomasum. The small intestinal mucosa showed villous atrophy. After training, the reticular groove reflex was re-established in a number of patients but the growth rate of these calves did not return to normal.

Enteric infections in veal calves: a longitudinal study on four veal calf units.

Forty-five calves on four veal calf units were monitored during the first four weeks after their arrival. Faecal samples were collected on alternate days and screened for the presence of rotaviruses, bovine coronavirus, Cryptosporidium oocysts, K99 positive strains of E. coli and Salmonella spp. Rotaviruses and Cryptosporidium were the most commonly detected agents (78% and 60% respectively of the calves). Bovine coronavirus was detected in the faeces of 18% of the calves, whilst K99 positive E. coli was only found in 2 samples from one calf. Salmonella spp. were not isolated from any of the 646 faecal samples examined. Shedding of rotaviruses occurred in a bimodal pattern beginning in the first week of the survey. Cryptosporidium oocysts were detected most frequently in the interval between the two peaks of rotavirus shedding. The presence of rotaviruses or Cryptosporidium oocysts in faeces was not strongly associated with scour, nor were combined infections with these agents or the cases of bovine coronavirus infection. The condition of the calves throughout the survey was generally satisfactory.

[Gentamicin: various pharmacotherapeutic aspects in comparison with other aminoglycosides]. / Gentamicine: enkele farmacotherapeutische aspecten met name in vergelijking met andere aminoglycosiden.

Gentamicin may be used in the treatment of infection with gram-negative bacteria including Pseudomonas spp and Proteus spp. Resistance will only appear in suboptimal or too prolonged courses of treatment and usually is due to 'multi-step mutation'. This resistance may be prevented, among others, by combined treatment with gentamicin and an antibiotic of the beta lactam group. When gentamicin is used correctly, it will have few toxic side-effects. Thus, 3 mg/kg of body weight three times daily will usually be indicated to ensure an optimum therapeutic effect. Parenteral administration of gentamicin would only appear to be useful in cases of bacteraemia and/or bacterial infection of the kidney and/or urinary excretory ducts; in the last-named case, the dose given at one time may be reduced by fifty per cent. Local treatment, the most recent method of which consists in administration by I(ntra-)T(racheal) route, apparently offers more prospects.

[Ruminal drinkers and clay defecators]. / Pensdrinkers en kleischijters.

In this clinical lecture, attention is drawn to the phenomenon of young milk-fed calves showing irregular appetites and recurrent bloat. These patients often produce faeces which are too dry and too light in colour. These symptoms are preceded by inadequate closing of the oesophageal groove during milk intake, which causes an inflow of milk into the rumen rather than into the abomasum. The present authors introduced the term 'Ruminal Drinkers' to describe this type of patient. The identification of ruminal drinkers as well as the clinical findings are reviewed. The effects of ruminal drinking, the therapy indicated and a number of preventive measures are discussed.