α5β1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers

Introduction

Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes.

Methods

All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes.

Results

In dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls.

Conclusions

The result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.     

Effects of Vascular Endothelial Growth Factor 165 on Bone Tissue Engineering

To study the relationship between vascular endothelial growth factor (VEGF) and formation and repair of engineering bone, second-generation bone marrow stromal cells (BMSCs) of New Zealand white rabbits that were separated in vitro were transfected with VEGF 165 gene vectors by adenovirus to detect gene expressions. Transfected BMSCs and β-tricalcium phosphate material were complexed and implanted at the femoral injury sites of the study group (n = 12), and the control group (n = 12) were implanted with engineering bones that were not transfected with VEGF. Femoral recoveries of the two groups were observed on the 15th, 30th, 45th and 60th days, and their vascularization and ossification statuses were observed by immunohistochemical methods. The BMSCs transfected with VEGF highly expressed VEGF genes and excreted VEGF. The two groups both experienced increased vascularization and bone volume after implantation (t = 7.92, P<0.05), and the increases of the study group were significantly higher than those of the control group (t = 6.92, P<0.05). VEGF is clinically applicable because it can accelerate the formation and repair of engineering bone by promoting vascularization and ossification.     

The Prevalence and Characteristics of Metabolic Syndrome in Patients with Vertigo

Objectives/Hypothesis

Metabolic syndrome (MetS) is a condition that increases the risk of coronary artery disease and cerebral infarction. We determined the prevalence of MetS in vertigo patients and clinically investigated the association between MetS and vertigo.

Study Design

Case-control study

Methods

The subjects were 333 patients, including 107 males and 226 females, who presented with vertigo as a primary symptom. MetS was diagnosed according to the International Diabetes Federation definition, which is based on waist circumference, blood serum levels, and blood pressure.

Results

MetS was detected in 53 (15.9%) of 333 vertigo patients, including 24 males (22.4%) and 29 females (12.8%); i.e., the frequency of MetS was significantly higher among the male patients than the female patients. The overall prevalence of MetS (15.9%) among vertigo patients did not differ from that observed among general adults in previous Japanese surveillance studies; however, MetS was significantly more common among the vertigo patients in males than general adult males. The prevalence of MetS was also examined in five types of vertigo, Concomitant MetS was noted in many males with vertebrobasilar insufficiency (VBI) and isolated vertigo of unknown etiology.

Conclusion

It was suggested that MetS is involved in the development of vertigo in males. MetS might be a risk factor for vascular vertigo such as VBI in males. The high frequency of MetS among males with vertigo of unknown etiology suggested that the pathogenesis of metabolic syndrome is involved in this type of isolated vertigo.     

Diversity of sulfur-cycle prokaryotes in freshwater lake sediments investigated using aprA as the functional marker gene

The diversity of sulfate-reducing prokaryotes (SRPs) and sulfur-oxidizing prokaryotes (SOPs) in freshwater lake ecosystems was investigated by cloning and sequencing of the aprA gene, which encodes for a key enzyme in dissimilatory sulfate reduction and sulfur oxidation. To understand their diversity better, the spatial distribution of aprA genes was investigated in sediments collected from six geographically distant lakes in Antarctica and Japan, including a hypersaline lake for comparison. The microbial community compositions of freshwater sediments and a hypersaline sediment showed notable differences. The clones affiliated with Desulfobacteraceae and Desulfobulbaceae were frequently detected in all freshwater lake sediments. The SOP community was mainly composed of four major phylogenetic groups. One of them formed a monophyletic cluster with a sulfur-oxidizing betaproteobacterium, Sulfuricella denitrificans, but the others were not assigned to specific genera. In addition, the AprA sequences, which were not clearly affiliated to either SRP or SOP lineages, dominated the libraries from four freshwater lake sediments. The results showed the wide distribution of some sulfur-cycle prokaryotes across geographical distances and supported the idea that metabolic flexibility is an important feature for SRP survival in low-sulfate environments.     

Ranging behavior of Mahale chimpanzees: a 16 year study

We have analyzed the ranging patterns of the Mimikire group (M group) of chimpanzees in the Mahale Mountains National Park, Tanzania. During 16 years, the chimpanzees moved over a total area of 25.2 or 27.4 km², as estimated by the grid-cell or minimum convex polygon (MCP) methods, respectively. Annually, the M group used an average of 18.4 km², or approximately 70 %, of the total home-range area. The chimpanzees had used 80 % of their total home range after 5 years and 95 % after 11 years. M group chimpanzees were observed more than half of the time in areas that composed only 15 % of their total home range. Thus, they typically moved over limited areas, visiting other parts of their range only occasionally. On average, the chimpanzees used 7.6 km² (in MCP) per month. Mean monthly range size was smallest at the end of the rainy season and largest at the end of the dry season, but there was much variability from year to year. The chimpanzees used many of the same areas every year when Saba comorensis fruits were abundant between August and January. In contrast, the chimpanzees used several different areas of their range in June. Here range overlap between years was relatively small. Over the 16 years of the study we found that the M group reduced their use of the northern part of their range and increased their frequency of visits to the eastern mountainous side of their home range. Changes in home-range size correlated positively with the number of adult females but not with the number of adult males. This finding does not support a prediction of the male-defended territory model proposed for some East African chimpanzee unit-groups.     

Biochemical Conversion of Uridine Diphosphate Glucose to Uridine Diphosphate 3-Ketoglucose by Washing Cells of Agrobacterium tumefaciens

The relation between the rate of increase in nonprotein nitrogenous compounds (NPN) of rabbit muscle and muscle pH ranging from 5.9 to 7.2 was examined during the post-mortem storage. Muscle of a high ultimate pH was prepared by the injection of ICH₂COOH into the vein. The more the muscle pH kept away from 6.3, the more NPN increased. Therefore, it has been suggested that the post-mortem proteolysis is mainly attributed to the acid proteolytic system comprising cathepsins in muscles at a pH lower than 6.3 and to the neutral proteolytic system in muscles at a pH higher than 6.3.

The ratio of the increment of ninhydrin positive materials to that of Cu-Folin phenol reagent positive materials among NPN was relatively large in muscles at a high pH. This result has suggested that the neutral proteolytic system was more abound in exopeptidase activity than acid proteolytic system.     

Purification and Properties of Maltose Phosphorylase from Lactobacillus brevis

Maltose phosphorylase (EC 2.4.1.8) from Lactobacillus brevis was purified 29-fold over the crude extract. The final preparation was at least 80% pure and had a specific activity of 18 units/mg protein. The molecular weights of the native enzyme and of the component dissociated in sodium dodecyl sulfate were 150,000 and 80,000, respectively. The enzyme does not contain pyridoxal-5′-phosphate as a cofactor. It can not act on maltitol, malto-triitol, sucrose, lactose and trehalose, and essentially not on isomaltose, maltobionic acid, maltotriose and maltotetraose. Inhibitory effect was observed with CuSO₄, HgCl₂ and p-chloromercuribenzoate. Some other properties were also examined. A possibility of using this enzyme for the analysis of maltose was proposed.     

Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B₁₂-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co²⁺-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co²⁺-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B₁₂ to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.

glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the presence of propionyl-CoA carboxylase in these bacteria, implies the participation of coenzyme B₁₂-dependent (R)-methylmalonyl-CoA mutase in the formation of succinate via the following route.

glutamate→α-ketoglutarate→α-hydroxyglutarate→propionate→propionyl-CoA→(S)-methylmalonyl-CoA→(R)-methylmalonyl-CoA→succinyl-CoA     

Bitter Taste of Synthetic C-Terminal Tetradecapeptide of Bovine β-Casein,H-Pro196-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val209-OH,and Its Related Peptides

The primary structure of bovine β-casein contains the partial sequence of -Pro¹⁹⁶-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val²⁰⁹ in the C-terminal portion. We previously reported that the synthetic C-terminal octapeptide, Arg²⁰²-Val²⁰⁹, is extremely bitter with its threshold value 0.004 mm, 250 times as strong as that of caffeine. To further investigate the bitter taste of the C-terminal portion of β-casein, we synthesized the C-terminal tetradecapeptide, Pro¹⁹⁶-Val²⁰⁹, and some of its fragments. A hydrophobic hexapeptide, Pro¹⁹⁶-Val²⁰¹, was twice as bitter as caffeine. The bitter taste of the decapeptide, Pro²⁰⁰-Val²⁰⁹, was the same as that of Arg²⁰²-Val²⁰⁹. Although the tetradecapeptide, Pro¹⁹⁶-Val²⁰⁹, was composed of two bitter peptides, Pro¹⁹⁶-Val²⁰¹ and Arg²⁰²-Val²⁰⁹, its bitter taste was weaker than that of Arg²⁰²-Val²⁰⁹ and its threshold value was 0.015 mm. We suggested that the increase of bitterness in peptides through the introduction of hydrophobic amino acids depended on the number of hydrophobic amino acids added. In addition, the synthetic retro analog of Arg²⁰²-Val²⁰⁹ (H-Val-Ile-Ile-Pro-Phe-Pro-Gly-Arg-OH) was not as bitter as Arg²⁰²-Val²⁰⁹. This indicated that the sequence of Arg²⁰²-Val²⁰⁹ is important for extreme bitterness.