Effect of mechanical stretching on expressions of muscle specific transcription factors MyoD, Myf-5, myogenin and MRF4 in proliferated myoblasts.

We examined expression of four important members of myogenic regulatory factors (MRFs) in the myoblasts both at mRNA and protein levels, which were subjected to mechanical stretching in in vitro condition. Our results showed that MyoD expression existed both in the stretch and in the control group at all time periods of the mechanical stimulus. Myf-5 expressed only at early stage of the stretch group. Although mRNA and protein expressions of myogenin and MRF4 were detected both in the stretch and in the control group at 12 h after the stretching, their expressions were only shown in the stretch group at 24 h after the mechanical stimulus. However, at 36 and 48 h, none of the MRFs examined except MyoD appeared in both groups. Our results suggest that the MRFs are up-regulated upon mechanical stimulus and each member plays a different major role for either proliferation or differentiation of the myoblasts.

Changes in the myosin heavy chain 2a and 2b isoforms of the anterior belly of the digastric muscle before and after weaning in mice.

During the process of growth and development, the digastric muscle is subjected to marked functional changes, including the change from suckling to mastication. In particular, because the anterior belly of the digastric muscle, which is one of the suprahyoid muscles, plays an important role in mastication. Therefore, this muscle seems to undergo a marked functional change before and after weaning. However, the details remain unknown. Here, to clarify the changes in the muscle fibre characteristics of the anterior belly of the digastric muscle before and after weaning, we examined myosin heavy chain isoforms at the protein (immunohistochemistry) and mRNA (transcription) levels. As a control, the changes in the muscle fibre characteristics of the sternohyoid muscle, which is anatomically aligned in the same direction as the anterior belly of the digastric muscle, were analyzed. The results showed that, in the anterior belly of the digastric muscle that is involved in mandibular movements in mice, the ratio of a fast-contraction isoform with strong contractile force increased after weaning. We believe that this occurred in response to a functional change from suckling to mastication. On the other hand, there was little change in the composition of sternohyoid muscle.

Changes in the properties of mouse tongue muscle fibres before and after weaning.

The purpose of this study was to clarify any changes in muscle fibre properties in different regions of murine tongue during development, and to assess the effects of functional changes including weaning on these muscle fibres. The tongue was divided into upper and lower regions at the lateral margin, and the expression of myosin heavy chain (MHC) isoforms at different ages was investigated. Expression of genes encoding MHC proteins was quantified at the transcription level by quantitative reverse transcriptase polymerase chain reaction, and the protein expression of MHC isoforms was assessed by immunostaining. No difference was found in isoform expression between the upper and lower regions of the tongue before weaning. However, the expression of MHC-2b increased markedly in both regions after weaning, while that of MHC-2a decreased. At the age of 16 weeks, the expression of MHC-2b in the lower region was greater than that in the upper region. These findings show that during weaning, when there is a shift from sucking behaviour to mastication, the expression of MHC-2b increases along with an increase in the speed and strength of muscle contraction. Also, contraction force becomes stronger in the lower region of the tongue than the upper region at the age of 16 weeks.

Superficial and deep layer muscle fibre properties of the mouse masseter before and after weaning.

To clarify changes in the properties of the masseter muscle superficial and deep layer muscle fibres, which initiate masticatory movement, myosin heavy chain isoforms were evaluated based on immunohistochemistry at the transcription level in male mice both before and after weaning. In the results, MHC-2b isoforms, the isoforms with the fastest contraction speed, were observed in the superficial layer after weaning. However, MHC-2a isoforms with slower contraction speeds were not apparent. By contrast, in the deep layer, MHC-2a isoforms were present, as were MHC-2b isoforms, however, there were fewer MHC-2b isoforms present than in the superficial layer. The most rapid movement in the mouse mandible was observed anteroposteriorly during mastication. As the superficial layer of the masseter muscle runs parallel to the direction of mandibular movement, the presence of MHC-2b isoforms in it is consistent. The presence of MHC-2a isoforms in the deep layer, lying at right angles to the direction of mastication movement, is consistent with the positional adjustment of the mandible contributed by the deep layer muscle fibres during masticatory movement. We therefore conclude that complicated masticatory movement is achieved by the presence of various muscle bundles within the masseter, each carrying out different roles.

[Recent features of parasites detected from clinical specimens].

It is generally considered that we have already been through with problems caused by various kinds of parasites which had once raged throughout the country. On the contrary to our common concept, we occasionally encounter some kinds of parasites in a laboratory as well as in clinical fields, which have become unfamiliar to us in these days. Parasitic diseases are in the first place, proper and limited to certain local regions, but the present situation has been greatly changed. Nowadays, specific parasites are no longer limited to polluted areas, but also they can be detected in any part of the world owing to facilitated transportations and a promoted international exchange of people. A recent gourmet boom is also one of the causes of infection as seen in anisakiasis. This study was conducted on the investigation of parasites detected from clinical specimens in our laboratory during the period from 1989 to 1993. 1) The following parasites were detected : (1) Strongyloides stercoralis, (2) Giardia lamblia, (3) Diphyllobothrium latum, (4) Schistosoma mansoni, (5) Entamoeba histolytica, (6) Necator americanus, (7) Isospora belli. 2) Strongyloides stercoralis was detected at the highest frequency. This result gives an account of high prevalence of the parasite among the inhabitants in Okinawa. In addition, the agar plate medium method which has been newly adopted has definitely led to far-advanced results for detection of this parasite. 3) Schistosoma mansoni and Necator americanus were found from foreigners one of whom was a Tanzanian and the other was a Dominican. 4) Isospora belli was found from those compromised cases such as ATL and AIDS.

Purification and characterization of a novel serine proteinase from the microsomal fraction of bovine pancreas.

A novel trypsin-like serine proteinase was purified to homogeneity from the bovine pancreas microsome fraction. The enzyme was solubilized with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), and purified by a series of column chromatographic steps on Ultrogel AcA-34, trypsin inhibitor-Sepharose 4B, and arginine-Sepharose 4B. The molecular mass of this pancreas trypsin-like proteinase (bPTLP) was estimated to be 29.5 kDa by SDS-PAGE under reducing conditions. The NH2-terminal sequence of bPTLP is very homologous, but not identical to those of other serine proteinases, especially such as elastases IV, II, and III. Substrate specificity studies involving a synthetic substrate and glucagon indicated that the enzyme hydrolyzes Arg-X, Lys-X, and Leu-X bonds. The best synthetic substrate for bPTLP was t-butyloxycarbonyl Gln-Arg-Arg-4-methylcoumaryl 7-amide. The enzyme failed to hydrolyze the substrate for chymotrypsin and elastase. The enzyme activity was inhibited by diisopropyl fluorophosphate, p-amidinophenylmethane sulfonylfluoride, and leupeptin, indicating that it is a serine-proteinase. These findings show that bPTLP is a novel serine-proteinase which differs from all known proteinases. The physiological function of the enzyme has yet to be determined.

Lysosomal enzyme replacement using alpha 2-macroglobulin as a transport vehicle.

Improvement of the delivery of exogenous enzymes is essential to achieve effective enzyme replacement therapy in lysosomal storage diseases. To test whether alpha 2-macroglobulin, an endogenous plasma protein, could serve as a transport vehicle of therapeutic agents to cells, alpha 2-macroglobulin and acid alpha-glucosidase or alpha-galactosidase A were coupled using two heterobifunctional cross-linking reagents. The alpha-glucosidase-alpha 2-macroglobulin conjugate was internalized and transported into lysosomes of acid alpha-glucosidase-deficient fibroblasts. The enzyme activity was stable after being taken up by the cells. Uptake of the conjugate resulted in the degradation of glycogen accumulated in lysosomes. The alpha-galactosidase A-alpha 2-macroglobulin conjugate was also internalized into the lysosomes of alpha-galactosidase A-deficient fibroblasts. Internalized alpha-galactosidase A-conjugate degraded globotriaosylceramide accumulated in lysosomes. The endocytosis of both conjugate was inhibited by alpha 2-macroglobulin-trypsin complex, indicating that the conjugates were endocytosed by an alpha 2-macroglobulin receptor system. These results showed the usefulness of alpha 2-macroglobulin as a transport vehicle of lysosomal enzymes for effective enzyme replacement.

Is the prevalence of HTLV-1 infection higher in Strongyloides carriers than in non-carriers?

To assess the concomitance of strongyloidiasis and HTLV-1 infection, an epidemiological survey was conducted in Okinawa, Japan, using the agar-plate culture, a highly sensitive method for detection of Strongyloides stercoralis. No significant difference in the positive rate of anti-HTLV-1 antibody was found between Strongyloides carriers and non-carriers. This result suggests that these two infections occur independently.