Turnover of cell-surface macromolecules in cultured dog tracheal epithelial cells

We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO4 for short periods (5-15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5-10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [3H]serine into the protein moiety of trypsin-accessible macromolecules occurred more slowly; trypsin-accessible 3H-labeled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool (t1/2 = 32 min). During continuous exposure to 35SO4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated (t1/2 = 7.5 h). After pulse-labeling the cells with 35SO4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface (t1/2 = 4.6 h), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4 degrees C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.     

Transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae has transglycosylation activity. Treatment of (Man)6(GlcNAc)2Asn with the enzyme in the presence of free N-acetylglucosamine gave a mixture of (Man)6GlcNAc and (Man)6GlcNAc beta 1----4GlcNAc mixture. N-Acetylglucosamine at the reducing end of the latter sugar chain was found by HPLC of the carbohydrate composition and of an exoglycosidase digest of the pyridylamino derivative of the reducing-end residue, and by 400 MHz 1H-NMR spectroscopy.     

Immunohistochemical distribution of CD9, heparin binding epidermal growth factor-like growth factor, and integrin alpha3beta1 in normal human tissues.

The tetra-membrane-spanning protein CD9 forms a complex with a membrane-anchored heparin binding epidermal growth factor-like growth factor (HB-EGF) and integrin alpha3beta1 in some human and monkey cell lines. We show here the immunohistochemical distribution of CD9, HB-EGF, and integrin alpha3beta1 in normal human tissues. Distribution of CD9, HB-EGF, and integrin alpha3beta1 was similar in various tissues, including transitional epithelium, squamous epithelium, thyroid follicular epithelium, adrenal cortex, testis, smooth muscle, and stromal fibrous tissue. However, distribution of the three proteins did not coincide in some tissues, such as lung, liver, kidney, gastric and intestinal epithelium, pancreas, salivary gland, and ovary. In striated muscle, including cardiac muscle, CD9 was present not in the muscle cells themselves but in the endomysium and perimysium, whereas HB-EGF was distributed in the muscle cells themselves. CD9 was distributed in the myelin, but HB-EGF was found in the axon of the peripheral and central nervous systems. Coincident distribution of integrin alpha3beta1 with others was not observed in muscles and neural tissues. In conclusion, there is a possibility of complex formation and functional cooperation of CD9 with HB-EGF and/or integrin alpha3beta1 in several tissues.     

Stochasticity of Intranuclear Biochemical Reaction Processes Controls the Final Decision of Cell Fate Associated with DNA Damage

A massive integrative mathematical model of DNA double-strand break (DSB) generation, DSB repair system, p53 signaling network, and apoptosis induction pathway was constructed to explore the dominant factors of unknown criteria of cell fate decision. In the proposed model, intranuclear reactions were modeled as stochastic processes and cytoplasmic reactions as deterministic processes, and both reaction sets were simulated simultaneously. The simulated results at the single-cell level showed that the model generated several sustained oscillations (pulses) of p53, Mdm2, ATM, and Wip1, and cell-to-cell variability in the number of p53 pulses depended on IR intensity. In cell populations, the model generated damped p53 oscillations, and IR intensity affected the amplitude of the first p53 oscillation. Cells were then subjected to the same IR dose exhibiting apoptosis induction variability. These simulated results are in quantitative agreement with major biological findings observed in human breast cancer epithelial MCF7, NIH3T3, and fibrosarcoma cells, demonstrating that the proposed model was concededly biologically appropriate. Statistical analysis of the simulated results shows that the generation of multiple p53 pulses is a prerequisite for apoptosis induction. Furthermore, cells exhibited considerable individual variability in p53 dynamics, which correlated with intrinsic apoptosis induction. The simulated results based on the proposed model demonstrated that the stochasticity of intranuclear biochemical reaction processes controls the final decision of cell fate associated with DNA damage. Applying stochastic simulation to an exploration of intranuclear biochemical reaction processes is indispensable in enhancing the understanding of the dynamic characteristics of biological multi-layered systems of higher organisms.     

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P < 0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P > 0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P < 0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P > 0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.