Trans-Epithelial Transport,Metabolism, and Biological Activity Assessment of the Multi-Target Lupin Peptide LILPKHSDAD (P5) and Its Metabolite LPKHSDAD (P5-Met)

P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.     

Adaptation of canine femoral head articular cartilage to long distance running exercise in young beagles.

OBJECTIVE--To study the effects of long term (one year), long distance (up to 40 km/day) running on the metabolism of articular cartilage the biosynthesis of proteoglycans was examined by in vitro labelling of anterior (weight bearing) and posterior (less weight bearing) areas of the femoral head from young beagles. METHODS--Total sulphate incorporation rates were determined and distribution of the incorporated sulphate was localised by quantitative autoradiography. Concentration and extractability of the proteoglycans were determined, and proteoglycan structures were investigated by gel filtration chromatography, agarose gel electrophoresis, and chemical determinations. RESULTS--In the less weight bearing area the amount of extractable proteoglycans was decreased (p < or = 0.02), simultaneously with an increased concentration of residual glycosaminoglycans in the tissue after 4 M GuCl extraction (p < or = 0.05). In control animals proteoglycan synthesis was most active in the deep zone of the cartilage, whereas exercise increased synthesis in the intermediate zone. There was a tendency to a lower keratan: chondroitin sulphate ratio in the running dogs. No macroscopical or microscopical signs of articular degeneration or injury were visible in any of the animals. CONCLUSION--The articular cartilage of the femoral head showed a great capacity to adapt to the increased mechanical loading. The reduced proteoglycan extractability in the less weight bearing area changed it similar to the weight bearing area, suggesting that the low extractability of proteoglycans reflects the long term loading history of articular cartilage. The congruency of the femoral head with acetabulum seems to protect the cartilage from the untoward alterations that occur in the femoral condyles subjected to a similar running programme.     

Analysis of lapine cartilage matrix after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate

OBJECTIVE: To study the short and long term effects of radiosynovectomy on articular cartilage in growing and mature rabbits. METHODS: The articular cartilage of the distal femurs of rabbits was examined four days, two months, and one year after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate ([(166)Ho]FHMA). Arthritic changes were evaluated from histological sections by conventional and polarised light microscopy, and glycosaminoglycan measurements using safranin O staining, digital densitometry, and uronic acid determination. Proteoglycan synthesis was studied by metabolic [(35)]sulphate labelling followed by autoradiography, and electrophoretic analysis of extracted proteoglycans. Northern analyses were performed to determine the mRNA levels of type II collagen, aggrecan, and Sox9 in cartilage samples. RESULTS: Radiosynovectomy had no major effect on the histological appearance of articular cartilage in mature rabbits, whereas more fibrillation was seen in [(166)Ho]FHMA radiosynovectomised knee joints of growing rabbits two months after treatment, but not after one year. Radiosynovectomy did not cause changes in the glycosaminoglycan content of cartilage or in the synthesis or chemical structure of proteoglycans. No radiosynovectomy related changes were seen in the mRNA levels of type II collagen, whereas a transient down regulation of aggrecan and Sox9 mRNA levels was seen in young rabbits two months after [(166)Ho]FHMA radiosynovectomy. CONCLUSIONS: [(166)Ho]FHMA radiosynovectomy caused no obvious chondrocyte damage or osteoarthritic changes in mature rabbits, but in growing rabbits some transient radiation induced effects were seen--for example, mild cartilage fibrillation and down regulation of cartilage-specific genes.     

Human articular cartilage proteoglycans are not undersulfated in osteoarthritis

Chondroitin sulfate is the major constituent of cartilage. Inadequate sulfate availability results in the production of undersulfated proteoglycans. In osteoarthritis, there is a net loss of articular cartilage proteoglycans. Theoretically, it is possible that during the progress of disease undersulfated glycosaminoglycans are synthesized producing proteoglycans with poorer biological properties. In this study, we tested whether in early human osteoarthritic articular cartilage (Mankin's score of 2 and 3) or more advanced disease (Mankin's score over 3), there are proteoglycans that contain a higher relative amount of nonsulfated chondroitin disaccharide isomer in their chondroitin sulfate chains by analyzing the molar ratios of chondroitin sulfate disaccharide isoforms with fluorophore-assisted carbohydrate electrophoresis. Our results indicated that the nonsulfated disaccharide of chondroitin sulfate formed in average only 1-2% of the total chondroitin sulfate. More important, the molar ratio of nonsulfated disaccharide did not appear to be increased in the osteoarthritic articular cartilage. We conclude that undersulfation of articular cartilage proteoglycans is not present in the human osteoarthritic joint.     

Effect of human trabecular bone composition on its electrical properties

Mechanical properties of bone are determined not only by bone mineral density (BMD), but also by tissue trabecular structure and organic composition. Impedance spectroscopy has shown potential to diagnose trabecular bone BMD and strength, however, the relationships between organic composition and electrical and dielectric properties have not been systematically investigated. To investigate these issues organic composition of 26 human trabecular bone samples harvested from the distal femur and proximal tibia was determined and compared with relative permittivity, loss factor, conductivity, phase angle, specific impedance and dissipation factor measured at wide range (50 Hz to 5 MHz) of frequencies. A strong linear correlation was found between the relative permittivity at 1.2 MHz and trabecular bone fat content (r = -0.85, p<0.01, n=26). On the other hand, relative permittivity measured at 200 Hz served as a good predictor of water content (r = 0.83). Phase angle, specific impedance and especially conductivity were strongly related to the trabecular bone dry density and water content (|r| > or = 0.69). Variation in bone tissue collagen content was strongly related to the relative permittivity measured at 1.2 MHz (r = 0.64), but only moderately to other parameters. Glycosaminoglycan content showed no significant relations with any investigated electrical parameters. The present study indicates that if the trabecular bone composition is known, the relationships presented in this study could facilitate calculation of current field distribution, e.g. during electrical stimulation of osteogenesis. On the other hand, our results suggest that permittivity measured at low (<1 kHz) or high (>100 kHz) frequencies could be used, e.g. during implant surgery, for prediction of trabecular bone water or fat contents, respectively.     

Seasonal variation of diagnosis of Type 1 diabetes mellitus in children worldwide

Aims To determine if there is a worldwide seasonal pattern in the clinical onset of Type 1 diabetes. Methods Analysis of the seasonality in diagnosis of Type 1 diabetes was based on the incidence data in 0‐ to 14‐year‐old children collected by the World Health Organization Diabetes Mondiale (WHO DiaMond) Project over the period 1990–1999. One hundred and five centres from 53 countries worldwide provided enough data for the seasonality analysis. The incidence seasonality patterns were also determined for age‐ and sex‐specific groups. Results Forty‐two out of 105 centres exhibited significant seasonality in the incidence of Type 1 diabetes (P < 0.05). The existence of significant seasonal patterns correlated with higher level of incidence and of the average yearly counts. The correlation disappeared after adjustment for latitude. Twenty‐eight of those centres had peaks in October to January and 33 had troughs in June to August. Two out of the four centres with significant seasonality in the southern hemisphere demonstrated a different pattern with a peak in July to September and a trough in January to March. Conclusions The seasonality of the incidence of Type 1 diabetes mellitus in children under 15 years of age is a real phenomenon, as was reported previously and as is now demonstrated by this large standardized study. The seasonality pattern appears to be dependent on the geographical position, at least as far as the northern/southern hemisphere dichotomy is concerned. However, more data are needed on the populations living below the 30th parallel north in order to complete the picture.     

Energy intake deficit and physical performance at altitude

BACKGROUND: Physical performance of sea-level (SL) residents acutely exposed to altitude (ALT) is diminished and may improve somewhat with ALT acclimatization. HYPOTHESIS: A large reduction in lean body mass (LBM), due to severe energy intake deficit during the first 21 d of ALT (4300 m) acclimatization, will adversely affect performance. METHODS: At ALT, 10 men received a deficit (DEF) of 1500 kcal x d(-1) below body weight (BW) maintenance requirements and 7 men received adequate (ADQ) kcal x d(-1) to maintain BW. Performance was assessed by: 1) maximal oxygen uptake (VO2max); 2) time to complete 50 cycles of a lift and carry task (L+C); 3) number of one-arm elbow flexions (10% BW at 22 flexions x min(-1); and 4) adductor pollicis (AP) muscle strength and endurance time (repeated 5-s static contractions at 50% of maximal force followed by 5-s rest, to exhaustion). Performance and body composition (using BW and circumference measures) were determined at SL and at ALT on days 2 through 21. RESULTS: At SL, there were no between-group differences (p > 0.05) for any of the performance measures. From SL to day 21 at ALT, BW and LBM declined by 6.6 +/- 3 kg and 4.6 kg, respectively, for the DEF group (both p < 0.01), but did not change (both p > 0.05) for the ADQ group. Performance changes from day 2 or 3 to day 20 or 21 at ALT were as follows (values are means +/- SD): VO2max (ml x min(-1)): DEF = +97 +/- 237, ADQ = +159 +/- 156; L + C (s): DEF = -62 +/- 35*, ADQ = -35 +/- 20* (*p < 0.05; improved from day 3); arm flex (reps): DEF = -2 +/- 7, ADQ = +2 +/- 8; AP endurance (min): DEF = +1.4 +/- 2, ADQ = + 1.9 +/- 2; AP strength (kg): DEF = -0.7 +/- 4, ADQ = -1.2 +/- 2. There were no differences in performance between groups. CONCLUSIONS: A significant BW and LBM loss due to underfeeding during the first 21 d of ALT acclimatization does not impair physical performance at ALT.