Effects of the masticatory demand on the rat mandibular development.

The influence of masticatory loading stimulus on mandibular development is not fully clear. In this paper, experimental alterations in the daily muscle use, caused by a changed diet consistency, were continuously monitored, while adaptations in bone and cartilage were examined. It is hypothesised that decreased muscular loading will result in a decrease in the growth factor expression and mandible growth. Fourteen 21-day-old Wistar strain male rats were randomly divided into two groups and fed on either a hard or soft diet for 14 weeks. An implanted radio-telemetric device recorded continuously muscle activity of the superficial masseter muscle. Chondroblast proliferation in the condylar cartilage was identified by insulin-like growth factor-1 receptor (IGF-1r) immunostaining. Furthermore, an X-ray was taken for cephalometric analysis. In the soft-diet group, the duty time of the superficial masseter muscle at higher activity levels was significantly lower than that in the hard-diet group. This decrease in muscular loading of the jaw system was accompanied by: a significant reduction in (i) articular cartilage thickness, (ii) expression of IGF-1r immunopositive cells and (iii) mandible ramus height. In conclusion, a decrease in masticatory demand during the growth period leads to insufficient mandibular development.

Changes in activity and structure of jaw muscles in Parkinson's disease model rats.

Parkinson's disease (PD), a major neurological disease, is characterised by a marked loss of dopaminergic neurons in the substantia nigra. Patients with PD frequently show chewing and swallowing dysfunctions, but little is known about the characteristics of their stomatognathic functions. The purpose of this study was to evaluate the influence of PD on jaw muscle fibre and functions. PD model rats were made by means of the injection of 6-hydroxydopamine (6-OHDA) into the striatum of 8-week-old Sprague-Dawley male rats. Five weeks after the injection, a radio-telemetric device was implanted to record muscle activity continuously from the superficial masseter and anterior belly of digastric muscles. Muscle activity was recorded for 3 days and was evaluated by the total duration of muscle activity per day (duty time). After recording the muscle activities, jaw muscles were isolated for immunohistochemical and PCR analyses. In PD model rats, the following findings of the digastrics muscles verify that compared to the control group: (i) the higher duty time exceeding 5% of the peak activity level, (ii) the higher expression of the mRNA of myosin heavy chain type I, and (iii) the tendency for fast to slow fibre-type transition. With respect to the masseter muscle, there were no significant differences in all analyses. In conclusion, PD leads to the changes in the jaw behaviours, resulting in a PD-specific chewing and swallowing dysfunctions.

Replicative epithelial cell cultures from normal human prostate gland.

Epithelial cell cultures of the normal human prostate gland were established. The subculturing of these cultures was accomplished with a novel nonenzymatic technique. These cultures were defined as normal epithelial cells on the basis of ultrastructure, karyotype, and inability to grow in soft agar.

Chromosomal analysis of human prostatic adenocarcinoma cell lines.

Although detailed cytogenetic analysis has been carried out in many types of cancer, there is little information on the chromosomal makeup of prostatic cancer cells. Karyological analyses of cell lines derived from both metastatic and primary prostatic carcinoma have been carried out by Q-, C-, and sequential banding techniques. The metastatic line, PC-3, isolated from a bone marrow specimen, is an established epithelial line which is tumorigenic in nude, athymic mice and forms colonies in semisolid agar suspension. A subline, PC-3/M, was isolated from a PC-3-induced mouse tumor. Karyotypic analysis of PC-3 by Q- and C-banding showed the cells to be aneuploid at all culture passage levels. The modal chromosome number shifted from 62 to 55 between the 5th and 50th passages. PC-3 has a unique karyotype. Chromosomes 2, 3, 5, 15, and Y were always absent. At least 11 different marker chromosomes were observed. The subline, PC-3/M, had a similar karyotype and retained the parental PC-3 markers. PC-3/M had a more restricted chromosomal frequency distribution range. Nearly 73% of the PC-3/M cells examined had 60 or 61 chromosomes in contrast to the wide distribution seen in PC-3. Silver staining for nucleolus organizer regions indicated that the number of functional nucleolus organizer regions in PC-3 was proportional to the number of acrocentric chromosomes. Banding analysis of PC-5-PI isolated from primary prostatic adenocarcinoma indicated that this line also had a characteristic karyotype with 28% pseudodiploid and 72% pseudotetraploid components. All metaphases examined were partially trisomic in chromosome 9 and lacked a demonstrable Y chromosome.

Development of tumorigenicity in simian virus 40-immortalized human bronchial epithelial cell lines.

Of five SV40-transformed clonal human bronchial epithelial cell lines previously shown to be nontumorigenic at early passages (R. R. Reddel et al., Cancer Res., 48: 1904-1909, 1988), two lines (BES-1A1 and BEAS-2B) from different donors have become weakly tumorigenic with further passaging. BES-1A1 passage 26 cells formed tumors in 3 of 9 athymic nude mice given s.c. injections, whereas BEAS-2B cells of > or = 32 passages formed highly cystic tumors at 8 of 58 injection sites after long latency periods [17 +/- 7 (SD) weeks]. These tumors took a total of 36 +/- 8 weeks to reach a diameter of 1.0 cm. Tumor cell lines were established from four BEAS-2B tumors, and these are resistant to the growth-inhibitory effects of serum, an inducer of squamous differentiation in BEAS-2B and normal bronchial epithelial cells. This finding supports the hypothesis that development of resistance to inducers of terminal squamous differentiation may be a step in the process of bronchial carcinogenesis. One of these tumor cell lines, B39-TL, is significantly more tumorigenic than the others and has a deletion from the short arm of chromosome 3 as has been described previously for some naturally occurring human bronchial carcinomas. Thus, from the clonally derived BEAS-2B cell line, cell populations with various degrees of tumorigenicity have developed. Analysis of the changes in these cells may yield insights into the multiple events involved in acquisition of the tumorigenic phenotype.

The brainstem and thalamic lesions in dentatorubral-pallidoluysian atrophy: an MRI study.

We studied the frequency and characteristics of brainstem and thalamic lesions in dentatorubral-pallidoluysian atrophy using MRI. Of 15 subjects diagnosed by DNA analysis, 13 had lesions in the pontine base, nine in the midbrain, and five in the thalamus. Lesions were correlated positively with the patient's age, but not with neurologic features or numbers of CAG repeats. Patients with Machado-Joseph disease or spinocerebellar ataxia 1 did not show these characteristic lesions.

Biochemical and cytogenetic studies of human lung cancers.

In ongoing studies, we have tested resected lung cancers from 41 men and 49 women; of those with primary lung cancer, 46 patients are free of disease and 35 have died of cancer or have persistent disease. Measurements and studies were as follows: total cellular deoxyribonucleic acid content by image analysis (n = 77); total genomic deoxyribonucleic acid methylation state and banding patterns from probed Southern blots (n = 36); radioimmunoassay for motilin, bombesin, gastrin, vasoactive intestinal peptide, and cholecystokinin (n = 18); and cytogenetic analysis (n = 39). All lung cancers were hyperploid. Adenocarcinomas and epidermoid carcinomas were generally hexaploid to nearly septaploid; comparisons by stage and histologic features suggested potential prognostic correlations. There was general hypomethylation of deoxyribonucleic acid (p less than 0.001). Deoxyribonucleic acid digests from restriction endonuclease Hpa II, when probed with deoxyribonucleic acid homologous to KPN, showed banding patterns that separated histologically indistinguishable primary adenocarcinomas and metastatic adenocarcinomas from one another. Cancers studied with radioimmunoassay were all negative for polypeptide hormones. Five cancers grew adequately in vitro to permit study of 190 detailed karyotypes (20 to 50 per tumor). Chromosome modal numbers ranged from 49 to 109. There were from 4 to 20 clearly abnormal marker chromosomes per tumor; abnormality derived from chromosome 1 was prevalent. Ten of 19 tumors xenotransplanted to nude mice were carried through two to five transplant generations without a change in histologic patterns.

Chromosomal changes and progressive tumorigenesis of human bronchial epithelial cell lines.

A simian virus 40 (SV40)-transformed human bronchial epithelial cell line, BEAS-2B, underwent progressive changes, including the development of tumorigenicity, during extended in vitro passaging. Karyotypic changes occurred in parallel with the phenotypic changes. For the first 12 passages following viral transformation, there were random karyotypic changes. Immortalization occurred between passages 12 and 21, corresponding with the accumulation of four characteristic abnormal chromosomes-m-1: add(15)(p11.1); m-2: der(8;9)(q10;q10); m-3: add(16)(p13); and m-4: mar4- and the loss of one homolog of chromosomes 8, 15, 16, 21, and 22. With further passaging (from 21 to 63), the acquisition of weak tumorigenicity was observed, accompanied by an increased frequency of cells containing all four common abnormal chromosomes, m-1 through m-4, and missing one normal homolog of chromosomes 8, 15, 16, and 22. Four tumor cell lines (B39-TL, B39-TR, B61-T4 and B61-T7) were established from tumors induced by the injection of these weakly tumorigenic BEAS-2B 39th- and 61st- passage cells into athymic nude mice. One of the cell lines, B39-TL, is significantly more tumorigenic than the others. It is notable that B39-TL showed two specific abnormal chromosomes, del(3p);der(3;15) (q10;q10) and m-6; der(21)t(3;21)(p14.2;p12) inducing deletion of a short arm of chromosome 3. Fluorescence in situ hybridization analysis with a probe for protein tyrosine phosphatase-gamma demonstrated loss of heterozygosity in the 3p14 region. The development of step-wise karyotypic changes in this in vitro carcinogenesis model parallels changes documented in several common human cancers.

Delayed embryonic development of mouse masseter muscle correlates with delayed MyoD family expression.

While the masseter muscle is known to have several unique developmental characteristics as compared with other skeletal muscles, little is known about its myogenesis. Thus, we examined the expression of myogenic marker and of myoD family gene mRNA from embryonic day (E) 11 to birth. The obtained results were compared with our earlier results of the mouse tongue muscle, which is also involved in oral functions. The mRNA quantities were determined by means of the reverse-transcription and competitive-polymerase chain-reaction techniques. The expression of myogenic marker mRNA indicated that differentiation and maturation in the masseter began at E13 as in the tongue, and were not yet completed at birth, although they were completed in the tongue. The expression of myoD, myogenin, and myf5 mRNA peaked later in the masseter (E17) than in the tongue (E13). The expression of MRF4 mRNA began later in the masseter (E15) than in the tongue (E13). These results suggest that the delayed expression of the myoD family genes in the masseter correlates with delayed differentiation and maturation, probably due to the later functional requirements of the masseter than of the tongue.

Developmental changes in the nicotinic acetylcholine receptor in mouse tongue striated muscle.

There are no published studies on synaptogenesis focusing on the elimination of the superfluous nicotinic acetylcholine receptor (nAChR) outside the neuromuscular junction and the nAChR subunit switch from the embryonic-type (alpha2betagammadelta subunits) to the adult-type (alpha2betaepsilondelta subunits) in mouse tongues. To identify the time course of nAChR subunit elimination and switch, we analyzed the expression levels of alpha, epsilon, and gamma subunit mRNAs, and the immunolocalization of the delta subunit protein in the mouse tongue and corresponding hind limb. The analysis included the period from embryonic day (E) 11 to the newborn stage. The nAChR elimination and subunit switch began at E15 in the tongue and at E17 in the hind limb. They were nearly complete at birth in the tongue, but not in the hind limb. The early completion of synaptogenesis in the tongue at birth may be related to the early functional demands placed on the tongue, such as suckling and swallowing, immediately after birth.

MRI findings of VIIth cranial nerve involvement in sarcoidosis.

Right facial nerve palsy in a 58-year-old woman was due to sarcoidosis demonstrated by Gd-DTPA enhanced MRI. Abnormal enhancement of the right VIIth cranial nerve in the distal internal acoustic canal was seen on MRI. The enhancing lesion was smaller after 1 month of prednisolone 50 mg day-1. This is the first report on facial nerve involvement in neurosarcoidosis examined by Gd-DTPA enhanced MRI. The use of Gd-DTPA enhanced MRI with thin slicing, e.g. 3 mm slice thickness and 1 mm interslice gap, is effective in detecting small extramedullary lesions.

Effect of an increase in occlusal vertical dimension on the rate of cyclic actin-myosin interaction in guinea-pig masseter muscle.

To study the effects of increased occlusal vertical dimension on these kinetics, the actin-filament sliding velocity on masseter myosins in an in vitro motility assay and the ATPase activity of masseter myosins from normal (control) and bite-opened (5.6 mm increase in the vertical dimension for 1 week) guinea-pigs were measured. In control myosin preparations, the average value (mean +/- SD, n = 5) for the actin-filament sliding velocity at 25 degrees C was 4.0 +/- 0.3 microns/sec. In bite-opened myosin preparations (n = 5), it was 3.4 +/- 0.3 microns/sec, a significant (p < 0.01) decrease. Myosin ATPase activity was also decreased significantly (p < 0.01) from 1.0 +/- 0.1 to 0.7 +/- 0.1 mumol Pi mg per min (mean +/- SD, n = 5) after the bite opening. These results strongly suggest that in guinea-pigs an increase in occlusal vertical dimension for 1 week decreases the turnover rate of actin-myosin interaction in the masseter through changes in the myosin isozyme. These changes may result in a slowing of the rate of detachment of myosin cross-bridges from actin filaments.

Quantitative changes in the mRNA for contractile proteins and metabolic enzymes in masseter muscle of bite-opened rats.

To study the effects of bite opening on the fibre phenotypes of rat masseter, the mRNAs of four predominant myosin heavy-chain isoforms (MHC I, IIa, IId/x and IIb) and two alkali light-chain isoforms (LC1f and 3f) as well as those of two metabolic enzymes, carbonic anhydrase III (CAIII, oxidative enzyme) and glucose-phosphate isomerase (GPI, glycolytic enzyme), were measured in relation to the total RNA of masseter muscle by competitive, reverse transcriptase-polymerase chain reaction in control and bite-opened rats. Bite opening (2.8 mm increase in the vertical dimension for 1 week) significantly (P<0.05) increased the amount of MHC IIa mRNA but decreased (P<0.001) the amount of MHC IIb mRNA without changing the amount of MHC IId/x mRNA. No MHC I mRNA was found in any masseter studied. A significant (P<0.01) increase in the mRNA of LC1f associated with a decrease (P<0.05) in that of LC3f was observed after the bite opening. The CAIII mRNA increased significantly (P<0.001), while the GPI mRNA decreased (P<0.05) in association with the bite opening. These results strongly suggest that in 1 week of bite opening changes the rat masseter muscle from a glycolytic, MHC IIb-LC3f-dominant fibre to an oxidative, MHC IIa-LC1f-dominant fibre.

Effects of diet consistency on the myosin heavy chain mRNAs of rat masseter muscle during postnatal development.

To study the effects of diet consistency on the fiber phenotypes of rat masseter (1-70 days of age), the mRNAs of myosin heavy chain isoforms (MHC embryonic, neonatal, I, IIa, IId/x and IIb) were measured in total RNA preparations from masseters of hard-diet group (HDG) and soft-diet group (SDG) by competitive reverse transcriptase-polymerase chain reaction (RT-PCR). With respect to the time course of the transition of each MHC mRNA expressed as a percentage relative to the maximum mean, the soft diet facilitated early (9 days after weaning) expression of IId/x and IIb isoforms, and also a decline in the expression of neonatal and IIa isoforms. The expression of neonatal, IIa and IId/x isoforms at 70 days of age was significantly (P<0.05, P<0.01, P<0.01, respectively) lower in SDG than in HDG, indicating a higher relative composition of the IIb isoform in the SDG. Embryonic MHC mRNA had disappeared by 14 days of age (i.e. before weaning at 19 days). No MHC I mRNA was observed in any masseter studied. These results suggest that in the rat a soft diet facilitates an even more MHC IIb-rich phenotype in the masseter muscle than a hard diet.

Adaptation of guinea-pig superficial masseter muscle to an increase in occlusal vertical dimension.

To study the effect of increased occlusal vertical dimension on the fibre phenotypes of the superficial masseter muscle, the composition of myosin heavy-chains (MHC), myosin light-chains (MLC) and tropomyosin was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in conjunction with densitometric analysis in normal (control) and bite-opened (5.7 mm increase in the vertical dimension for 1 week) guinea-pigs. The superficial masseter contained two fast-type MHC isoforms, II-1 and II-2, in both the bite-opened and control groups; their relative content (mean+/-SD, n = 7) was 47.8+/-2.9% and 52.2+/-2.9%, in the bite-opened and 44.4+/-3.0% and 55.6+3.0% in control preparations, indicating no significant (p>0.05) changes in MHC composition in association with the bite opening. On the other hand, significant differences in MLC and tropomyosin composition were found between the two preparations. Although the MLC consisted of three components, LC1f, LC2f and LC3f, in both preparations, their relative content (mean+/-SD, n = 7) was 37.1+/-2.4%, 49.6+/-1.6% and 13.2+/-3.2%, respectively, in the bite-opened and 28.1+/-3.1%, 50.9+/-1.6% and 21.0+/-3.5% in the control preparations, indicating that the bite opening induced a significant (p < 0.0001) increase in the relative content of LC1f at the expense of that of LC3f. Although the tropomyosin consisted of two components, TM-alpha and TM-beta, in both preparations, their relative content (mean+/-SD, n = 7) was 91.8%+/-1.9% and 8.2+/-1.9%, respectively, in the bite-opened and 95.9+/-0.7% and 4.1+/-0.7% in the control preparations, showing a significant (p < 0.001) increase in the relative content of TM-beta in relation to the bite opening. These results indicate that in guinea-pigs an increase in occlusal vertical dimension for 1 week changes the composition of MLC and tropomyosin, with no significant change in MHC, in the masseter muscle. These changes might be required to meet altered functional demands.

Expression of myogenic regulatory factors during the development of mouse tongue striated muscle.

While the role of myogenic regulatory factors (MRFs) in skeletal myogenesis has been well evaluated in limb and trunk muscles, very little is known about their role in tongue myogenesis. Here the expression of MRF mRNA in mouse tongue muscle was examined during development from embryonic day (E)11 to birth and compared them with that in hind-limb muscle. Desmin, muscle creatine kinase and troponin C mRNAs were used as markers for myoblast determination, myotubule formation and myofibre maturation, respectively. The mRNA quantities were determined by competitive reverse transcriptase-polymerase chain reaction. The expression profile of desmin mRNA indicated that myoblast determination occurred before E11 in both the tongue and hind-limb muscles; the profile of muscle creatine kinase and troponin C mRNAs indicated that myotubule formation and myofibre maturation began between E11 and 13 in both tongue and hind-limb muscles, but ended 2 days earlier in the tongue than in the hind limb. Expression of myoD and myogenin mRNAs began at E11, increased, and showed peak values earlier in the tongue muscle (E13) than in the hind-limb muscle (E15). Expression of MRF4 mRNA appeared earlier in the tongue (E13) than in the hind-limb muscle (E15) and increased in both muscles after that. These results suggest that myotubule formation and myofibre maturation in the tongue muscle progress faster than in the hind-limb muscle, a result of earlier expression of myoD, myogenin, and MRF4 in response to earlier functional demands such as suckling immediately after birth.

Postnatal changes in the nicotinic acetylcholine receptor subunits in rat masseter muscle.

No published study on synaptogenesis in masseter muscle has focused on the shift of nicotinic acetylcholine receptors (nAChRs) from the embryonic type (alpha(2)-, beta-, gamma- and delta-subunits) to the adult-type (alpha(2)-, beta-, epsilon- and delta-subunits) and the elimination of nAChRs outside the neuromuscular junction. To identify the time course of the nAChR transitions in rat masseter muscle between 1 and 63 days of age, the expression of delta-, epsilon- and gamma-subunit mRNAs was analysed by competitive polymerase chain reaction in combination with reverse transcription. The expression of the delta-subunit was high between 1 and 7 days of age, then decreased by 95% (P<0.0001) between 7 and 28 days, suggesting that the nAChR elimination occurs during this period. The quantity of the epsilon-subunit increased by approximately 600% (P<0.0001) between 1 and 21 days of age, whereas the quantity of the gamma-subunit decreased by 85% (P<0.0001) during the same period. This result indicates that the nAChR type shift is terminated at 21 days of age. The feeding behaviour of the rats inevitably changed from suckling to biting after 19 days of age, because they were weaned at that age. As the nAChR type shift was terminated soon after weaning, the termination could be related to the change in feeding behaviour. However, it might also be the case that nAChR elimination is not directly related to the change in feeding behaviour, as the elimination continued at the same rate for 9 days after weaning (from 19 to 28 days of age).

High performance liquid chromatographic separation of stereoisomeric bile acids as their UV-sensitive esters.

High performance liquid chromatographic separation of a series of mono-, di- and trihydroxylated 5 beta-cholanic acids, which differ only in position and configuration of hydroxyl groups at positions C-3, C-7 and/or C-12, is reported. The C-24 free acids were derivatized to four different classes of UV-sensitive esters, i.e., p-bromophenacyl (BP), m-methoxyphenacyl (MP), 4-nitrophthalimidemethyl (NPM) and 9-anthrylmethyl (AM) esters, and chromatographed on two variants of C18 reversed-phase columns (Nova-Pak C18 and Zorbax ODS) with methanol-water systems as mobile phase. Separation efficiency and elution order of some isomeric pairs were influenced by both the structure of the C-24 ester groups and the nature of the columns used. Excellent chromatographic properties were found for those derivatives, particularly for the NPM esters.

Fall in forearm skin temperature during grade walking on a treadmill.

Thermoregulatory responses were observed in 7 male subjects during grade walking at different speeds of between 0.5 and 9 km/hr. The total heat productions (H) in uphill and downhill walking are defined as metabolic heat production (M)--external work (W) and M + W, respectively. During 60 min exercise at 20 degrees C, r. h. 40%, rectal temperature increased to levels dependent on M and independent of H. The sweat-rate varied in proportion to H, and not to M. During 10 min exercise, the environmental conditions (28 degrees C, 40% r.h.) and work intensities were set so that changes in skin temperature could be observed without the interference of sweating. The results indicated that the fall in forearm skin temperature was correlated to M, and not to H. Our previous studies showed that cutaneous vasoconstriction persisted during exercise and that raised work intensities increase the surface area affected by lowered skin temperature. These findings suggest that the rise in core temperature during exercise results from the decreased dry heat loss due to a fall in skin temperature.

Fall in skin temperature during exercise observed by thermography.

Monochromatic and colored thermographies were used to visualize skin temperatures during exercise without involvement of sweating. Skin temperature began to fall immediately at the onset of exercise, remained low during exercise and rose rapidly after cessation of exercise. Increased work intensities produced a proportional fall in skin temperature and greater surface area of lowered temperature.