Effect of L-3,4-dehydroproline on collagen synthesis and prolyl hydroxylase activity in mammalian cell cultures.

The incorporation of DL-3,4-dehydro[14C]proline into collagen and total protein of 3T3 cells occurred at approximately one-fifth the rate observed for L-[14C]proline. Addition of L-3,4-dehydroproline to the culture medium inhibited markedly the incorporation of [14C]glycine and L-[3H]lysine into the collagen of 3T3 cells, but there was only slight inhibition of the incorporation of the radiolabeled amino acids into total cellular proteins, indicating that the action of L-3,4-dehydroproline is specific for collagen. When 1 mM L-3,4-dehydroproline was added to the culture medium the [14C]hydroxyproline content was reduced 40% in the cell layer and 70% in the medium. The D isomer of 3,4-dehydroproline did not inhibit [14C]hydroxyproline formation. These findings indicate that L-3,4-dehydroline reduced the hydroxylation of the susceptible prolyl residues in the collagen molecule and the secretion of collagen from the cell. The reduction in the hydroxyproline content is probably related in part to a reduction in the activity of prolyl hydroxylase; when various mammalian cell cultures were exposed to 0.2 mM L-3,4-dehydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroxylase was not affected. Under these conditions, cell growth and lactic dehydrogenase required protein synthesis. Removal of L-3,4-dehydroproline from the growth medium resulted in a time-dependent increase in the specific activity of prolyl hydroxylase.     

N-乙酰半胱氨酸对大鼠心脏成纤维细胞增殖和胶原合成的影响

目的:通过观察N-乙酰半胱氨酸(NAC)对大鼠心脏成纤维细胞(CFs)增殖和胶原合成的影响,探讨NAC对心脏重构的作用。方法:以培养的新生SD大鼠CFs为实验对象,给予不同浓度的NAC进行干预,48小时后用MTT比色法检测CFs增殖水平,用3H脯氨酸掺入法测定总胶原合成。结果:与对照组相比,不同浓度NAC作用下的CFs增殖水平和3H脯氨酸掺入量均比对照组低,且具有浓度依赖性(p<0.05)。结论:NAC能够抑制SD大鼠CFs增殖,并降低其胶原合成,因此NAC对心脏的病理性重构可能具有保护作用。     

N-乙酰半胱氨酸对大鼠心脏成纤维细胞增殖和胶原合成的影响

目的：通过观察N-乙酰半胱氨酸（NAC）对大鼠心脏成纤维细胞（CFs）增殖和胶原合成的影响,探讨NAC对心脏重构的作用。方法：以培养的新生SD大鼠CFs为实验对象,给予不同浓度的NAC进行干预,48小时后用MTT比色法检测CFs增殖水平,用3H脯氨酸掺入法测定总胶原合成。结果：与对照组相比,不同浓度NAC作用下的CFs增殖水平和3H脯氨酸掺入量均比对照组低,且具有浓度依赖性（p〈0.05）。结论：NAC能够抑制SD大鼠CFs增殖,并降低其胶原合成,因此NAC对心脏的病理性重构可能具有保护作用。     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Effect of beta-D-xylosides on collagen synthesis in cultured fibroblasts.

Cultured normal human skin fibroblasts were incubated with [¹⁴C]proline in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose. Formation of non-dialyzable hydroxyproline was used as a measure of collagen synthesis. Although total [¹⁴C]proline incorporation was similar in the two cultures, [¹⁴C]hydroxyproline formation was significantly decreased in the β-xyloside-treated cultures. Increasing the period of incubation increased the radioactivity of the insoluble collagen fraction in untreated fibroblasts, however, in β-xyloside-treated cultures no such increase was observed. In contrast to the decreased production of collagen, growth of cells in the presence of the β-xyloside induced the synthesis of high levels of soluble glycosaminoglycans as measured by ³⁵SO₄ incorporation into isolated polysaccharide.     

Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

Background  

Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.%) of N.     

Effect of ascorbate on collagen synthesis by lung embryonic fibroblasts

Summary Total insoluble collagen and hydroxyproline formation were examined in lung embryonic fibroblasts (IMR-90) grown in the presence or absence of added ascorbate. As expected, when the cells from both groups (+ and −ascorbate) are pulsed with [¹⁴C]proline in the presence of ascorbate, the percent hydroxylation in a 24-hr period does not vary significantly. However, there are dramatic differences in the quantity and quality of the insoluble collagen fraction produced by those cells grown for a long period of time with added ascorbate. Those cells deprived of continuous addition of ascorbate to the culture medium do not display large quantities of accumulated collagen in the cell layer fractions as measured by the hydroxyproline content, whereas the cells grown in the presence of ascorbate contain significant amounts of accumulated collagen. A new method for examining the extracellular insoluble collagen produced in cell cultures is described in these studies. With the aid of pancreatic elastase relatively pure insoluble collagen can be obtained from cells grown in culture. In those cells grown in the presence of ascorbate, the purified insoluble collagen yeilds appropriately banded fibrils when examined in the electron microscope and has an amino-acid composition that is compatible with pure collagen. On the other hand, those cells grown in the absence of ascorbate do not yield purified insoluble collagen as determined by these same criteria. The elastase procedure for the purification of insoluble collagen in cell cultures is simple, easy to use and allows one to assess additional aspects of collagen biosynthesis.     

Collagen synthesis and deposition during mammary epithelial cell spreading on collagen gels

Mouse mammary epithelial cultivated on collagen gels demonstrate active spreading as the cells form monolayers. In this novel system, initiation of cell spreading is preceded by de novo synthesis of type IV collagen. The newly synthesized collagen is partitioned such that after 48 hr, approximately 24% is found in the culture medium, 35% is intracellular, and 41% is deposited in the extracellular matrix of the developing epithelium. Cultures deprived of serum failed to spread and to synthesize collagen. Proline analogues were shown to inhibit cell spreading and to suppress collagen synthesis in a dose-dependent manner. Cytochalasin D inhibition of F-actin elongation was shown to prevent cell spreading but not to suppress total collagen synthesis. During cytochalasin D treatment, inhibition of cell spreading was shown to result from failure to deposit or to maintain deposited collagen in the epithelium extracellular matrix. The data indicate that synthesis and extracellular deposition of a major basal lamina component (viz. type IV collagen) must precede and then accompany epithelial cell spreading in collagen gel culture. It is suggested that the microfilament apparatus, through some hypothetical integral membrane protein, can anchor extracellular type IV collagen, which then provides a necessary condition for cell spreading.     

Effect of growth hormone on fibrinogen synthesis

A method described by McFarlane (1963a) for the measurement of the absolute rate of synthesis of liver-made plasma proteins was used to show that, within a few hours of giving bovine growth hormone to rats, fibrinogen synthesis increased significantly without a change in albumin synthesis.     

Regulation of cell wall synthesis and growth

The replication and growth of Mycobacterium tuberculosis are fundamentally linked to the synthesis and extension of its complex cell wall. Incorporation of new wall material must be tightly regulated so that its deposition does not compromise the extant structure. M. tuberculosis also produces an impressive array of complex bioactive lipids that are intimately involved in pathogenesis and protective immunity. The profiles of these lipids are regulated appropriately to allow the bacterium to respond to the prevailing conditions it faces in vivo. A number of regulatory strategies employed by M. tuberculosis to control cell wall biosynthesis and cell division have now been elucidated. The review highlights the role of alternative sigma factors with extracytoplasmic function in the activation of genes for biosynthesis of complex lipids involved in pathogenicity. Rel(Mtb) and CRP(Mt) play roles in cell wall responses to general nutrient deprivation by synthesis and sensing of starvation second messengers, respectively. Recently, the importance of protein phosphorylation networks in cell wall biosynthesis has attracted considerable interest. A plethora of two-component and eukaryotic-like serine/threonine protein kinases systems have been discovered and several are implicated in cell-division, morphogenesis and regulation of the profile of complex bioactive lipids elaborated by the pathogen.     

Effect of reinnervation on collagen synthesis in rat skeletal muscle.

The effect of reinnervation on the activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), both enzymes of collagen biosynthesis, and on the concentration of hydroxyproline (Hyp) was studied in gastrocnemius, soleus, and tibialis anterior muscles of rat 19, 26, 40, and 61 days after crush denervation of the sciatic nerve. The GGT activity was elevated in denervated gastrocnemius and soleus muscles and the PH activity in gastrocnemius. Muscular Hyp concentration was increased in denervated tibialis anterior muscle. Both the PH and GGT activities and the Hyp concentration returned to the control level during the reinnervation period (19-61 days from the start of denervation). It seems that denervation atrophy of skeletal muscle is associated with an increased rate of muscular collagen biosynthesis and that during reinnervation collagen synthesis rate decreases despite accelerated muscular growth. The results thus suggest that innervation is a powerful suppressive regulator of muscular collagen biosynthesis.     

Effect of phase of growth and hyperlipidemic serum on the synthesis of collagen in rabbit aortic smooth muscle cells in culture

The aim of this study was to elucidate wheter long-term cultivation in the presence of hyperlipidemic serum is able to induce changes in the rate of synthesis of collagen and other proteins by arterial smooth muscle cells. Rabbit aortic medial cells were grown in 10% sera and their collagen and total protein synthesis were studied by incubation of the cells with radioactive proline. When the cells were grown in fetal calf serum, their collagen synthesis was low after trypsinization but reached a constant level in one week, whereafter it remained within 4--5% of total protein synthesis for up to 30 days. Cultivation in hyperlipidemic rabbit serum for up to 14 days caused an accumulation of lipid droplets in the cells, but there were no detectable changes in the rate of collagen of total protein synthesis when compared with cells grown in normal rabbit serum.     

Effect of cardiac lymph flow obstruction on cardiac collagen synthesis and interstitial fibrosis

The effect of chronic cardiac lymphatic obstruction on the myocardial synthesis of collagen type I and III was investigated in a rabbit model. In the lymphatic obstruction group (n=16), plasma C-terminal propeptide type I procollagen (PICP) and N-terminal propeptide type III procollagen (PIIINP) were elevated at 7, 14 and 30 days after the operation (p<0.05). The elevated PICP and PIIINP returned to the pre-operation values 60 days after the operation. The myocardial expression of collagen type I and III mRNA were also enhanced in the lymphatic flow obstruction group. Plasma PICP, PIIINP and myocardial collagen type I and III mRNA remained unchanged in the control group (n=16). We concluded that chronic obstruction of cardiac lymph flow leads to enhanced myocardial collagen synthesis in rabbits. The enhanced collagen synthesis starts within seven days after lymphatic obstruction and subsides after 60 days.     

Immobilization of growth factors on collagen scaffolds mediated by polyanionic collagen mimetic peptides and its effect on endothelial cell morphogenesis

Angiogenesis, a morphogenic event endothelial cells (ECs) undergo in response to 3-D environmental triggers, is critical to the survival and ultimate functional capacity of engineered tissue constructs. Here we present a new collagen mimetic peptide (CMP) architecture consisting of multiple anionic charges at the peptide's N-terminus designed to attract growth factors by charge-charge interactions and bind to collagen by CMP-collagen interaction. The anionic CMPs exhibited specific binding affinity to type I collagen substrates while attracting vascular endothelial growth factors (VEGFs), which led to enhanced morphological features of ECs, indicative of tubulogenesis. The results show that these new CMPs could be used to direct proliferation and differentiation of cells in collagen scaffolds by localization and sustained delivery of growth factors and other morphogens.