Biomedical subjects

M H Lu

Publications and source records attributed to M H Lu.

At least 19 recordsLinked to original sources

PubMed2006

Ultrasound palpation sensor for tissue thickness and elasticity measurement--assessment of transverse carpal ligament.

Palpation is a traditional diagnostic procedure for health care professionals to use their fingers to touch and feel the body soft tissues. It is a common clinical approach, though it is rather subjective and qualitative and the palpation results may vary among different people. Tissue ultrasound palpation sensor (TUPS) provides a feasible solution that makes the palpation of soft tissues not subjective feeling any more. It is comprised of an ultrasound transducer together with a load cell to form the finger-sized probe. The probe is used to push against the soft tissue surface to measure the thickness and elasticity of the soft tissues. TUPS has been successfully applied to the assessment of various human tissues. Recently, we have improved TUPS, which can now be linked to personal computer (PC) via universal serial bus (USB) and provide a better user-interface. The information of ultrasound signal and indentation force is displayed on PC in real time during measurement. In this paper, we introduce the recent application of TUPS for the assessment of the transverse carpal ligament. The tissues at the carpal tunnel regions of five normal male subjects were tested using TUPS. The results showed that the average thickness of the tissues covering the carpal tunnel ligament and the tunnel region was 7.98+/-1.05 mm and 9.59+/-1.12 mm, respectively. Under a compression force of 20 N applied by a cylindrical ultrasound indentor with a diameter of 9 mm, the stiffness of the soft tissue layer and the tunnel region was 6.72+/-2.10 N/mm and 15.63+/-8.42 N/mm, respectively. It is expected that TUPS can be a potential tool for non-invasive assessment of carpal tunnel syndrome.

Algorithms↗

PubMed2005

Development of a portable 3D ultrasound imaging system for musculoskeletal tissues.

3D ultrasound is a promising imaging modality for clinical diagnosis and treatment monitoring. Its cost is relatively low in comparison with CT and MRI, no intensive training and radiation protection is required for its operation, and its hardware is movable and can potentially be portable. In this study, we developed a portable freehand 3D ultrasound imaging system for the assessment of musculoskeletal body parts. A portable ultrasound scanner was used to obtain real-time B-mode ultrasound images of musculoskeletal tissues and an electromagnetic spatial sensor was fixed on the ultrasound probe to acquire the position and orientation of the images. The images were digitized with a video digitization device and displayed with its orientation and position synchronized in real-time with the data obtained by the spatial sensor. A program was developed for volume reconstruction, visualization, segmentation and measurement using Visual C++ and Visualization toolkits (VTK) software. A 2D Gaussian filter and a Median filter were implemented to improve the quality of the B-scan images collected by the portable ultrasound scanner. An improved distance-weighted grid-mapping algorithm was proposed for volume reconstruction. Temporal calibrations were conducted to correct the delay between the collections of images and spatial data. Spatial calibrations were performed using a cross-wire phantom. The system accuracy was validated by one cylinder and two cuboid phantoms made of silicone. The average errors for distance measurement in three orthogonal directions in comparison with micrometer measurement were 0.06+/-0.39, -0.27+/-0.27, and 0.33+/-0.39 mm, respectively. The average error for volume measurement was -0.18%+/-5.44% for the three phantoms. The system has been successfully used to obtain the volume images of a fetus phantom, the fingers and forearms of human subjects. For a typical volume with 126 x 103 x 109 voxels, the 3D image could be reconstructed from 258 B-scans (640 x 480 pixels) within one minute using a portable PC with Pentium IV 2.4 GHz CPU and 512 MB memories. It is believed that such a portable volume imaging system will have many applications in the assessment of musculoskeletal tissues because of its easy accessibility.

Equipment Design↗

PubMed2005

A novel noncontact ultrasound indentation system for measurement of tissue material properties using water jet compression.

This study is aimed to develop a novel noncontact ultrasonic indentation system for measuring quantitative mechanical properties of soft tissues, which are increasingly important for tissue assessment and characterization. The key idea of this method is to use a water jet as an indenter to compress the soft tissue while at the same time as a medium for an ultrasound beam to propagate through. The use of water jet indentation does not require a rigid compressor in front of the focused high frequency ultrasound transducer to compress the tissue, so that the additional attenuation caused by the rigid compressor and the strong echoes reflected from its surfaces can be avoided. The indentation deformation was estimated from the ultrasound echoes using a cross-correlation algorithm and the indentation force was calculated from the water pressure measured inside the water pipe. Experiments were performed on uniform tissue-mimicking phantoms with different stiffness. The Young's moduli and Poisson's ratios of these phantoms were measured using a uniaxial ultrasound compression system. The ratio of the indentation pressure to the tissue relative deformation was obtained from the water indentation. This ratio was well correlated with the Young's modulus (r = 0.87). The results also demonstrated that the water indentation approach could differentiate materials with different stiffness in a combined phantom (288 kPa and 433 kPa). This novel noncontact water indentation approach could be potentially used for the measurement of the elasticity of small samples and with a fast scanning speed.

Algorithms↗

PubMed2004

High resolution ultrasound elastomicroscopy imaging of soft tissues: system development and feasibility.

Research in elasticity imaging typically relies on 1-10 MHz ultrasound. Elasticity imaging at these frequencies can provide strain maps with a resolution in the order of millimetres, but this is not sufficient for applications to skin, articular cartilage or other fine structures. We developed a prototype high resolution elastomicroscopy system consisting of a 50 MHz ultrasound backscatter microscope system and a calibrated compression device using a load cell to measure the pressure applied to the specimen, which was installed between a rigidly fixed face-plate and a specimen platform. Radiofrequency data were acquired in a B-scan format (10 mm wide x 3 mm deep) in specimens of mouse skin and bovine patellar cartilage. The scanning resolution along the B-scan plane direction was 50 microm, and the ultrasound signals were digitized at 500 MHz to achieve a sensitivity better than 1 microm for the axial displacement measurement. Because of elevated attenuation of ultrasound at high frequencies, special consideration was necessary to design a face-plate permitting efficient ultrasound transmission into the specimen and relative uniformity of the compression. Best results were obtained using a thin plastic film to cover a specially shaped slit in the face-plate. Local tissue strain maps were constructed by applying a cross-correlation tracking method to signals obtained at the same site at different compression levels. The speed of sound in the tissue specimen (1589.8+/-7.8 m s(-1) for cartilage and 1532.4+/-4.4 m s(-1) for skin) was simultaneously measured during the compression test. Preliminary results demonstrated that this ultrasound elastomicroscopy technique was able to map deformations of the skin and articular cartilage specimens to high resolution, in the order of 50 microm. This system can also be potentially used for the assessment of other biological tissues, bioengineered tissues or biomaterials with fine structures.

Animals↗

PubMed2004

Indentation test of soft tissues with curved substrates: a finite element study.

Indentation is a commonly used approach to measure the mechanical properties of soft tissues, such as articular cartilage and limb tissues. The Young's modulus of tissue can be calculated from the indentation test using a mechanical model, where the soft tissue is normally assumed to have a flat substrate. In this study, a series of 2D finite element models were established to investigate the effects of bones with various curvatures embedded in the soft tissues during an indentation test. For each curvature of the hard substrate, the errors in the calculation of the Young's modulus were estimated for different indentation depths (0-10%) and aspect ratios a/h of the indentor diameter and the tissue thickness (0.2-2 in seven steps). The radius ratio a/R of the indentor and the curved substrate ranged from 0 to 0.38 in nine steps. Results showed that the error in calculation of the Young's modulus increased by 21.2% when the curvature a/R of the bone increased from 0 to 0.38 (under the condition of a/h=1.0, Poisson's ratio v=0.45). The error increased from 6.0 to 18.2% when the tissue thickness increased from 0.2 to 2 (a/R=0.18, v=0.45). It was found that the error in the Young's modulus calculation caused by the curved hard substrates could be corrected by a single factor for different indentation depths. This factor depends on the Poisson's ratio, the aspect ratio a/h and the radius ratio a/R.

Bone and Bones↗

PubMed2001

[Inhibitory effect of the combination of DHPAs and hyperbaric oxygen on Japanese B encephalitis virus infection in mice].

OBJECTIVE: To investigate the inhibitory effect of the combination of 2,3-dihydroxypropyl-adenine (DHPAs) and hyperbaric oxygen (HBO) on Japanese B encephalitis virus infection in mice. METHODS: Mice infected P3-55 and P3-53 virus were used in the experiments. Virus was inoculated intraperitoneally to Kumming strain mice (8.5-9.5 g). Mice were divided into groups and treated in the incubation period of the infection. The duration of treatment was 5 days, and the experiments were terminated at the end of 2 weeks after treatment. The living and the dead were then counted finally. RESULTS: The intergroup comparison showed marked difference between the control group and combination of DHPAs and hyperbaric oxygen group (P < 0.025 and P < 0.01), but there was no statistical difference between other groups and the control group (P > 0.05). CONCLUSION: The combined treatment of (S) DHPA and/or (RS)-DHPA and HBO has inhibitory effect on Japanese encephalitis virus infection in experimental mice.

Adenine↗

PubMed2001

Effect of varying dietary fat levels on rat growth and oxidative DNA damage.

Dietary fat has previously been shown to have somewhat complicated relationships to levels of oxidative stress in rats. In this study, we examined the effects of five different dietary fat intakes on levels of oxidative DNA damage in rats. Animals fed diets containing 3%, 5%, 10%, or 15% corn oil had body weights that were similar after 20 weeks. Animals fed a 20% fat diet, however, had significantly higher mean body weight than any other group. Levels of 5-hydroxymethyl-2'-deoxyuridine, one marker of oxidative DNA damage, had different relationships to dietary fat in blood and mammary gland. In blood, levels increased with dietary fat levels, and the highest levels were observed with the 20% fat diet (65% higher levels than with the 3% fat diet). In mammary gland, a plateau-type effect was observed, with maximal levels of oxidative DNA damage being obtained using 10% fat (representing a 68% increase relative to the 3% fat diet). This could be a result of induction of compensatory mechanisms in response to a high-fat diet in mammary gland but not in the short-lived nucleated blood cells. Oxidative DNA damage levels in blood thus appear to be a marker of dietary fat intake. In mammary gland, however, levels of DNA damage are consistent with previously observed promotional effects of dietary fat on mammary gland tumorigenesis at lower levels of fat intake with little or no incremental promoting effects at higher levels of fat intake.

Animals↗

PubMed2001

Paracrine function of cultured aortic endothelial cells in spontaneously hypertensive rats.

BACKGROUND: Vasoconstrictor and vasodilator release from vascular endothelial cells not only regulates vascular tone but also induces vascular smooth muscle cell proliferation. METHODS: In order to understand the role of vasoconstrictor and vasodilator release in the development of hypertension in spontaneously hypertensive rats (SHR), aortic endothelial cells were isolated and cultured from 4-week-old and 24-week-old SHR (SHR-4 and SHR-24) and age-matched Wistar-Kyoto rats (WKY-4 and WKY-24) used as control. Prostacyclin (PGI2), endothelin-1 (ET-1) and thromboxane A2 (TXA2) release from cultured endothelial cells in the culture medium, were measured after 30 min with or without treatment with acetylcholine, calcium ionophore A23187 or thrombin. RESULTS: The results showed that there was no significant difference in ET-1 secretion between SHR-4 and age-matched WKY rats, but ET-1 secretion was about twice as high in SHR-24 as in WKY-24. TXA2 secretion was significantly higher in SHR-4 than in WKY-4 and was also higher than in SHR-24, but there was no significant difference between SHR-24 and WKY-24. The secretion of PGI2 was higher in SHR-24 than in WKY-24 and also higher than in SHR-4 and WKY-4. The prostaglandin PGI2 and TXB2 secretions from all groups of cultured VECs treated with various reagents, acetylcholine, calcium ionophore A23187 or thrombin were increased in similar patterns. However, there was no significantly different response between SHR and WKY VECs. CONCLUSIONS: Similar levels of ET-1 secreted from endothelial cells between SHR-4 and WKY-4 indicated that ET-1 secretion seems not a crucial factor in early hypertension development in SHR. The high level of TXA2 secretion in SHR-4 may involve in early hypertension development in SHR.

Animals↗

PubMed2000

Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte.

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.

Cell Differentiation↗

PubMed2000

Effects of single injection of methylazoxymethanol at postnatal day one on cell proliferation in different brain regions of male rats.

Methylazoxymethanol (MAM), an aglycone of cycasin extracted from Cycad seed, is reported to induce microencephaly in rats after prenatal or postnatal administration. Forty postnatal day (PND) 1 rats derived from 8 timed-pregnant rats were used to evaluate the effect of a single subcutaneous injection of MAM on PND 1 on cell proliferation in rat brain. All dams were fed NIH-31 diet. In the MAM-treated group, each pup received a single injection of MAM at 10 mg/kg body weight. In the control group, pups were injected with saline solution. Body weight of pups was recorded on PNDs 1, 7, 14, 21, 28, and 60. One male pup in each litter was removed for sacrifice by decapitation on PNDs 7, 14, 21, 28, and 60. The brain was removed and dissected to obtain the brain stem, caudate nucleus, cerebellum, frontal cortex, hippocampus, hypothalamus, and olfactory bulb. The percentage (%) of S-phase cells in the cell cycle for each region was measured as index of cell proliferation. A significant (p<0. 05) reduction in body weight was detected on PND 7. The % S-phase cells in cerebellum on PND 7 and olfactory bulb on PND 14 initially increased and then later decreased. There were significant decreases in the % S-phase cells in both cerebellum and caudate nucleus on PND 21 and olfactory bulb on PND 28. No significant changes were found in other brain regions in cell proliferation activity between the control and MAM-treated groups. The % of S-phase cells in cerebellum decreased 65% on PND 60 but lacked statistical significance due to small number of animals used in each group. The results indicated that a single injection of MAM at 10 mg/kg/body weight on PND 1 inhibited cell proliferation in the cerebellum in a manner that could lead to microencephaly.

Aging↗

PubMed1999

The effect of dietary fat on malondialdehyde concentrations in Fischer 344 rats.

The effects of dietary fat and age on the level of malondialdehyde (MDA), a product of lipid peroxidation, were investigated in cerebellum, kidney, and liver tissues of female Fischer 344 rats. Groups of rats were fed diets containing various levels of corn oil (3, 5, 10, 15, or 20%), starting at 57 days of age, for a duration of 2, 10, or 20 weeks. High fat diets are thought to promote tumor formation, diabetes, and cardiovascular diseases via induction of oxidation stress, and this can begin early in the lifespan. However, it was observed that rats chronically consuming 3 and 5% corn oil diets yielded significantly higher levels of MDA, as analyzed by high-performance liquid chromatography, compared with those fed higher fat diets. After 20 weeks of feeding, the concentration of MDA in each of the three organs studied showed no significant differences among rats consuming diets containing 10, 15, or 20% corn oil. The levels of MDA were highest in the cerebellum, followed by kidney, and lowest in liver. Over the 20-week feeding period, a decrease in MDA level in both cerebellum and liver was observed.

Aging↗

PubMed1999

[Relationship between the expression of p53 and C-erbB-2 oncoproteins and biologic behaviors of human mucoepidermal carcinoma of salivary gland]

OBJECTIVE:To study the relationship between the expression of p53 and c-erbB-2 proteins and biologic behaviors of human mucoepidermal carcinoma.METHODS:The expressions of oncogene proteins p53 and c-erbB-2 in 32 cases of human mucoepidermal carcinomas were investigated with microwave immunohistochemistry.RESULTS:p53 and c-erbB-2 oncoproteins in duct epithelial cells around cancer tissues were 10.0% and 15.0%,respectively,but those in the acinar cells affiliated with the salivary gland ducts were negative. The positive rates of p53 oncoprotein and c-erbB-2 oncoprotein in mucoepidermal carcinoma were 40.6% and 46.9%,respectively. Both p53 and c-erbB-2 oncoproteins were expressed in mucous cells,epidermoid cells and intermediate cells of mucoepidermal carcinoma. The expression of p53 and c-erbB-2 oncoproteins in human mucoepidermal carcinoma tissues were related to histological patterns,differentiation degree of cancer and tumor recurrence (P<0.01).CONCLUSION:This study suggested that both p53 and c-erbB-2 on coproteins,as the markers for differentiation of cancer,might be useful in screening progress and evaluating prognosis of patients with mucoepidermal carcinoma.

Journal Article↗

PubMed1998

The effect of food restriction on the composition of intestinal microflora in rats.

The effect of a food-restricted diet on the fecal microflora of rats was studied by determining total anaerobic bacteria, bacterial cellular fatty acids, and the predominant intestinal bacteria shown by polymerase chain reaction (PCR) primers specific for the 16S rRNA gene sequences of 12 bacterial species. Twenty-four female Fischer 344 rats, 57 days old were divided into two groups and maintained on an NIH-31 diet. One group was fed ad libitum while the other group received 60% of ad libitum food intake (40% food restriction supplemented with vitamins and minerals equal to the ad libitum animals). After 2, 10, and 20 weeks on this dietary regimen, groups of four animals were sacrificed and the intestinal contents analyzed for changes in the bacterial flora. The anaerobic population for two-week (short-term) food-restricted rats was 3.2 x 10(8) per gram, slightly less than the 9.1 x 10(8) per gram found in the ad libitum-fed rats. The anaerobic populations in 20-week food restricted and ad libitum fed rats were 1.9 x 10(9) and 2.7 x 10(9) per gram, respectively. The total anaerobic population did not change significantly in either group during the 20-week study. No statistically significant differences were observed in the bacterial cellular fatty acid profiles between the two groups as determined by gas-liquid chromatography. PCR analysis of the intestinal contents indicated no significant shifts in the predominant flora due to dietary changes. The results, using three different methods to detect changes in the rat intestinal microflora, suggest that long-term dietary restriction had little effect on the microflora of female Fischer 344 rats.

Animals↗

PubMed1998

The correlation of blood pressure, age and endothelin-1 binding sites in aortic smooth muscle cells of rats.

Spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD) rats at the ages of 4, 8, 12, 16 and 24 weeks were used to examine the effects of age on the density of endothelin-1 (ET-1) binding sites in aortic smooth muscle cells and systolic blood pressure (SBP). The SBP of the 3 different rat strains was measured, and the maximum binding value (Bmax) and dissociation constant (Kd) of ET-1 binding sites in smooth muscle cells of the thoracic aorta were determined. The results showed that the SBP and Bmax values of SHR, WKY and SD rats increased with age; the SBP and Bmax value at each corresponding age were significantly higher in SHR than in WKY and SD rats, however, there was no significant difference between WKY and SD rats. The relationship of age vs SBP, age vs Bmax, and Bmax vs SBP showed significantly positive correlation in all 3 rat strains. The regression line in the Bmax of endothelin binding sites against SBP in the 3 different rat strains presented a similar slope. These results indicate that SBP, which increased with age, could be related to an increased density of ET-1 binding sites on vascular smooth muscle cells in these 3 different rat strains.

Aging↗

PubMed1997

Comparison of endothelin binding sites in cultured aortic smooth muscle cells from spontaneously hypertensive and normotensive rats.

Endothelin-1 (ET-1), which is secreted from vascular endothelial cells, is not only a potent vasoconstrictor but also a vascular smooth muscle cell growth factor. The direct effect of ET-1 on vascular smooth muscle cells, mediated via its specific receptor may therefore play an important role in hypertension and atherosclerosis. Our previous studies indicated that ET-1 secretion from cultured aortic endothelial cells from spontaneously hypertensive rats (SHRs) at the prehypertensive stage (4 weeks old) was not significantly different from that of cells from age-matched Wistar-Kyoto (WKY) rats. In this study, the binding of ET-1 to cultured aortic smooth muscle cells from SHRs and WKY rats was studied. Using tissue explant techniques, rat aortic smooth muscle cells from SHRs and age-matched WKY rats of different ages (4 and 24 weeks old) were successfully cultured in vitro. The maximum binding capacity (Bmax) and binding affinity (Kd) of ET-1 to cultured aortic smooth muscle cells were evaluated by a receptor-binding assay. The data revealed that the affinity of ET-1 binding to smooth muscle cells was similar in all 4 groups of experimental rats. However, the Bmax of cultured smooth muscle cells from 24-week-old SHRs was 2.5 times higher than of smooth muscle cells from age-matched WKY rats (8.64 +/- 0.72 vs 3.69 +/- 0.10 fmol/10(6) cells) and 1.5 times higher than in aortic smooth muscle cells from 4-week-old SHRs (8.64 +/- 0.72 vs 5.36 +/- 0.36 fmol/10(6) cells). These results suggest that hypertension in SHRs may be related to a high density of ET-1 receptors on arterial smooth muscle cells.

Animals↗

PubMed1996

Triiodothyronine-induced hyperthyroidism and reproduction in female rats.

The effect of L-triiodothyronine (L-T3)-induced hyperthyroidism on reproduction in female rats was studied. Treatment groups used were normal control, 1, 2, 4, 8, and 16 micrograms L-T3/100 g body weight (b.w.)/day. Subcutaneous injections of L-T3 began the first day of estrus until day 1 or day 19 of lactation. All rats given 1, 2, or 4 micrograms L-T3 had regular estrous cycles and normal number of pregnancies. Levels of L-T3 at 8 or 16 micrograms results in abnormal estrous cycles. Only one pregnancy occurred in the 8 micrograms group and no rats became pregnant in the 16 micrograms group. While groups on 1 or 2 micrograms L-T3 had normal parturition, groups on 4 micrograms or more showed signs of dystocia. Only the 1 microgram L-T3 group lactated to day 20, but at 67% of normal control. All levels above 1 micrograms showed loss of pups by day 2 of lactation due to lack of milk production, loss of maternal behavior, and cannibalism. Mammary gland wet and dried fat-free tissue (DFFT) weights were decreased in all treated groups except 1 microgram L-T3. The wet and DFFT weights of thyroids and uteri on days 2 or 20 of lactation were not altered by L-T3 treatment, but levels above 1 microgram increased both wet and DFFT weights of adrenal glands and ovaries. The data indicate that the no observed adverse effect level (NOAEL) of L-T3 for female rat reproduction would be below 1 microgram/100 g b.w./day. L-T3 treatment at 1 microgram/100 b.w./day or above is detrimental. These effects may be mediated through a hormonal imbalance due to the accelerated metabolic rate in reproductive tissues associated with hyperthyroidism.

Adrenal Glands↗

PubMed1995

Isolation and cultivation of aortic endothelial cells from spontaneously hypertensive rat with a modified tissue explant technique.

In the present study, we successfully established a "tissue explant technique" to obtain high yield and purity of endothelial cells from the aorta of hypertensive and normotensive rats (SHR and WKY). Small pieces of aorta were placed on fibronectin precoated petri dishes. The effects of oxygenation in the tissue preparation stage, tilting of the petri dish during the explanting period and timing of the removal of tissue blocks from petri dishes were evaluated. These procedures appeared to be critical for cell survival, tissue adhesion and minimizing of non-endothelial cell contamination. The cultured endothelial cells were characterized by morphological, immunohistochemical and biochemical examination. The cultured cells from both SHR and WKY rats showed similar endothelial cell character, positive immunofluorescence staining for the von Willebrand factor, and uptake of acetylated low-density lipoprotein (DiI-ac-LDL). The secretory function of prostacylcin I2 (PGI2), thromboxane A2 and endothelin of cultured endothelial cells was measured. The results showed that the secretion of both PGI2 and endothelin was greater in SHR than in WKY rats, but that there was no difference in thromboxane A2 secretion. Therefore, our "tissue explant technique" can provide high yield and purity of endothelial cells with their specific biological function in vitro. It will permit us to further study the role of endothelial cells in the development of hypertension.

Animals↗

PubMed1994

Sequential appearance of muscle-specific proteins in myoblasts as a function of time after cell division: evidence for a conserved myoblast differentiation program in skeletal muscle.

Based on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle-specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0-24 h postmitosis) by video time-lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x-h-old to define their age relative to their mitotic "birthdate." All 6 h myoblasts that emerged in a mitogen-rich medium were desmin+ but only 50% were positive for a alpha-actin, troponin-I, alpha-actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up-regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were alpha-actinin+ or zeugmatin+ but MyHC- or titin- whereas others were troponin-I+ or MyHC+ but alpha-actinin- or alpha-actin-. In 15-h-old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle-shaped cells; and (2) the sarcolemma became fusion-competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP-C (C protein). The emergence of one, or a string of approximately 2 mu long sarcomeres, was invariably characterized by the localization of myomesin and MyBP-C to their mature positions in the developing A-bands. The latter group of A-band proteins may be rate-limiting in the assembly program. The great majority of myoblasts stained positively for desmin and myofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle-specific proteins in vitro fully recapitulates myofibrillar assembly steps in myoblasts of the myotome and limb bud in vivo, as well as in nonmuscle cells converted to myoblasts by MyoD. We suggest that this cell-autonomous myoblast differentiation program may be blocked at different control points in immortalized myogenic cell lines.

Animals↗