Net Protein Accumulation Research Articles

The abundant class III chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins.

Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening-associated proteins. Analyses of individual proteins revealed that a thaumatin-like protein, a beta-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas.

Proline accumulation in developing grapevine fruit occurs independently of changes in the levels of delta1-pyrroline-5-carboxylate synthetase mRNA or protein.

Mature fruit of grapevine (Vitis vinifera) contains unusually high levels of free proline (Pro; up to 24 micromol or 2.8 mg/g fresh weight). Pro accumulation does not occur uniformly throughout berry development but only during the last 4 to 6 weeks of ripening when both berry growth and net protein accumulation have ceased. In contrast, the steady-state levels of both the mRNA encoding V. vinifera Delta1-pyrroline-5-carboxylate synthetase (VVP5CS), a key regulatory enzyme in Pro biosynthesis, and its protein product remain relatively uniform throughout fruit development. In addition, the steady-state protein levels of Pro dehydrogenase, the first enzyme in Pro degradation, increased throughout early fruit development but thereafter remained relatively constant. The developmental accumulation of free Pro late in grape berry ripening is thus clearly distinct from the osmotic stress-induced accumulation of Pro in plants. It is not associated with either sustained increases in steady-state levels of P5CS mRNA or protein or a decrease in steady-state levels of Pro dehydrogenase protein, suggesting that other physiological factors are important for its regulation.

Embryonic salivary gland epithelial branching activity is experimentally independent of epithelial expansion activity

Embryonic mouse submandibular salivary gland rudiments undergo morphogenesis in organ culture, characterized by extensive epithelial growth and expansion and repetitive branching activity. Tunicamycin, at a concentration of 25 ng/ml culture medium, decreases the degree of net protein accumulation by 83% and the degree of epithelial expansion by 70% compared to controls, over a 48-hr culture tenure. These decreases correlate with reduced incorporation of [ 3H]thymidine into DNA. Nevertheless, epithelial branching activity is uncompromised, undergoing an ∼10-fold increase in lobe numbers, in both controls and tunicamycin-treated rudiments, during the same 48-hr period. The effect is most striking during the 24- to 48-hr culture interval, when controls and tunicamycin-treated rudiments each triple their lobe numbers and controls approximately double epithelial area, while tunicamycin virtually stops all epithelial expansion.

15N-tracer kinetic studies on the utilization of urea in protein metabolism of infants

The virtual importance of the urea circuit is not clear. After a 3 to 21 day application of 100 mg [15N]-urea/l in 15 infants a [15N]-excess value of 0.06 in serum protein could be proven. Taking as a basis a protein content of 11.4% of the body mass and a regular distribution of the [15N] within the body one can calculate a retention of the urea nitrogen in the protein pool of 40.4% of the intake. Taking in account an amount of 11.4% urea nitrogen from the total nitrogen in mother's milk then the amount of urea nitrogen from the net protein accumulation comes to 6.5 (3.1-11.8)%.

Microstructure and Biochemistry of Avian Muscle and its Relevance to Meat Processing Industries

This review is an attempt to summarize information concerning the gross and microscopic anatomy of avian muscle. In addition, specific proteins found in muscle tissue such as the troponins, tropomyosin, α- and β-actinin, desmin, vimentin, myomesin, and creatine kinase are described. The role of protein synthesis and degradation, leading to net protein accumulation in growing animals, is addressed. It is thought that learning to decrease the rate of protein degradation would have immense economic significance. Satellite cells are mentioned as some of these cells fuse with muscle cells to become true muscle nuclei. Understanding the mechanism by which the nuclei of satellite cells differentiate into muscle nuclei could lead to a practical means of increasing protein synthesis within muscle tissue. Finally, postmortem biochemistry is discussed to show how the pH of muscle tissue interacts with environmental temperature to affect the final tenderness of the product.

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03 % serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03 % serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.

Control of protein degradation and growth phase in normal and neoplastic cells.

Cells have to double their protein mass in order to divide. Whether this is achieved through increased synthesis (PS), decreased degradation (PD), or a combination of both is still debated. Likewise open are other basic questions: whether, beyond differences relating to growth phase (GP) or rate, reduced PD rates are a general characteristic of neoplastic versus normal cells, conferring to them a definite growth advantage; which mechanisms are operating the PD regulation, if any, during GP transitions, and which ones may be defective in neoplastic cells. Growing liver under conditions of regeneration or development is known to achieve a net protein accumulation thanks to increased PS, and particularly, to decreased PD rates, as compared with the adult, steady-state tissue; the level of lysosomal proteinase (LP) activities is reduced; in the regenerating liver this reduction has been located in cycling hepatocytes. AH-130 Yoshida ascites hepatoma cells effect the transition from log to stationary GP by concurrently reducing PS and accelerating PD (slow turnover protein pool); while PD is virtually not affected by lysosomal inhibitors (LI) in growing cells, the extra PD in resting cells is all inhibitable; there is no regulation of LP levels over this GP transition, which is due to depletion of oxygen and nutrients. GP transitions in normal 3T3 cells are also coupled with regulations of both PS and PD, the extra PD in quiescent cells being all suppressible by LI. Quiescence of 3T3 cells, due to depletion of growth factors, is associated with a marked elevation of some LP activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Progress through G1 and S in relation to net protein accumulation in human NHIK 3025 cells

We have investigated whether human NHIK 3025 cells are dependent upon a net increase in cellular protein content in order to traverse G1 and S. The increase in DNA and protein content was studied by means of two-parameter flow cytometry using populations of cells synchronized by mitotic selection. By adding 1 μM cycloheximide to the medium protein synthesis was partially inhibited, resulting in negligible net accumulation of protein. The cells were able to enter S and progress through S under such conditions. The latter was the case whether the cells had been accumulating protein during G1 or not. The results further indicate that the larger cells enter S earlier and traverse S at a higher rate than the smaller cells. Our conclusion is that net accumulation of protein does not seem to be a prerequisite for traverse through G1 and S, i.e. DNA replication may be dissociated from the general growth of cell mass.

Regulation of protein accumulation in cultured cells.

1. A technique is described whereby protein synthesis, protein breakdown and net protein accumulation are measured separately in monolayer cultures of mammalian cells. All rates are expressed as microgram of protein per 18 h incubation. 2. Under most incubation conditions with either L6 rat myoblasts or T47D human breast carcinoma cells the rates of protein accumulation, determined directly, agreed with the rates obtained by subtracting protein breakdown from protein synthesis. 3. Foetal calf serum, human and bovine colostrum, human milk and insulin increased protein accumulation in both cell lines, mainly as a consequence of effects on protein synthesis. 4. NH4Cl, in addition to inhibiting protein breakdown in both cell lines in the presence and in the absence of serum, stimulated protein synthesis in L6 myoblasts. 5. Leupeptin slightly inhibited protein breakdown without affecting protein-synthesis rates. 6. Cycloheximide almost completely inhibited protein synthesis, but restricted the net loss of cell proteins under most conditions because protein-breakdown rates were also decreased. 7. The assumptions, limitations and potential application of this technique for evaluating changes in protein turnover are described.

Protein turnover and proliferation. Failure of SV-3T3 cells to increase lysosomal proteinases, increase protein degradation and cease net protein accumulation.

The contrasting control of lysosomal proteinases, protein turnover and proliferation was studied in 3T3 and SV-3T3 (SV-40-virus-transformed 3T3) cells. 1. In 3T3 cells, net protein accumulation proceeded from 5%/h (doubling time, T(d)=14h) in growing cells to 0%/h as cells became quiescent. SV-3T3 cells never ceased to gain protein, but rather decreased their protein accumulation rate from 6-7%/h (T(d)=10-12h) to 2%/h (T(d)=35-40h) just before culture death in unchanged medium. 2. In both cell types the rates of protein synthesis per unit of protein (a) were proportional to the initial serum concentration from 0 to 6%, and (b) declined under progressive depletion of undefined serum growth factors. In depleted growth medium, leucine incorporation per unit of protein in 3T3 and SV-3T3 cells declined to almost equal synthetic rates while the 3T3 cell existed in a steady state of zero net gain, and the SV-3T3 cell continued to gain protein at a rate of 2%/h. 3. Whereas a large fraction of the control of 3T3-cell net protein accumulation can be accounted for by an increase in degradation from 1%/h to 3%/h, the SV-3T3 cell did not exhibit a growth-related increase in degradation appreciably above 1%/h. 4. Thus, by using first-order kinetics, the continued net protein accumulation of the transformed cell can be accounted for by a failure to increase protein degradation, whereas fractional synthesis can be made to decline to a rate similar to that in the quiescent non-transformed cell. 5. Upon acute serum deprivation, both cell types similarly exhibited small rapid increases in proteolysis independent of cell growth state or lysosomal enzyme status. 6. The 3T3 cell increased its lysosomal proteinase activity in conjunction with increase in the growth-state-dependent proteolytic mechanism; however, the SV-3T3 cell failed to increase lysosomal proteinases or the growth-state-dependent proteolytic mechanism.

Protein turnover and proliferation. Turnover kinetics associated with the elevation of 3T3-cell acid-proteinase activity and cessation of net protein gain.

1. At least 95% of the total protein of A31-3T3 cell cultures undergoes turnover. 2. First-order exponential kinetics were used to provide a crude approximation of averaged protein synthesis, Ks, degradation, Kd, and net accumulation, Ka, as cells ceased growth at near-confluent density in unchanged Dulbecco's medium containing 10% serum. The values of the relationship Ka = Ks - Kd were : 5%/h = 6%/h - 1%/h in growing cells, and 0%/h = 3%/h - 3%/h in steady-state resting cells. 3. As determined by comparison of the progress of protein synthesis and net protein accumulation, the time course of increase in protein degradation coincided with the onset of an increase in lysosomal proteinase activity and decrease in thymidine incorporation after approx. 2 days of exponential growth. 4. After acute serum deprivation, rapid increases in protein degradation of less than 1%/h could be superimposed on the prevailing degradation rate in either growing or resting cells. The results indicate that two proteolytic mechanisms can be distinguished on the basis of the kinetics of their alterations. A slow mechanism changes in relation to proliferative status and lysosomal enzyme elevation. A prompt mechanism, previously described by others, changes before changes in cell-cycle distribution or lysosomal proteinase activity. 5. When the serum concentration of growing cultures was decreased to 1% or 0.25%, then cessation of growth was accompanied by a lower steady-state protein turnover rate of 2.0%/h or 1.5%/h respectively. When growth ceased under conditions of overcrowded cultures, or severe nutrient insufficiency, protein turnover did not attain a final steady state, but declined continually into the death of the culture.

The relation between protein accumulation and cell cycle traverse of human NHIK 3025 cells in unbalanced growth.

Human NHIK 3025 cells, synchronized by mitotic selection, were given 2 mM thymidine, which inhibited DNA synthesis without reducing the rate of protein accumulation. After removal of the thymidine the cells proceeded towards mitosis and cell division, with an S duration 2 hours shorter than, but a G2 and M duration nearly identical to that of the control cells. If cycloheximide (1.25 muM) was present together with thymidine, no net protein accumulation took place during the treatment, and the subsequent duration of S, G2, and M was similar to that of untreated cells. The shortening of S seen after treatment with thymidine alone would therefore indicate that the rate of DNA synthesis depended on the amount of some preaccumulated protein. The postreplicative period in thymidine-treated cells was lengthened by cycloheximide treatment although the protein content had already been doubled. This suggests that proteins required for the traverse of this part of the cell cycle might have to be synthesized after completion of DNA replication. Shortly after removal of thymidine, the rate of protein accumulation declined markedly, indicating the existence of some mechanism for negative control of cell mass. In addition, the daughters of thymidine-treated cells had their cell cycle shortened by 2 hours. As a result, the cells had returned to balanced growth already in the first cell cycle following the induction of unbalanced growth. In conclusion, our experiments suggest that NHIK 3025 cells might require a minimum time in order to traverse the cell cycle, which is independent of cell mass.

Protein synthesis and degradation during expression of the temperature-sensitive defect in ts A1S9 mouse L-cells.

The involvement of altered protein metabolism in the expression of the temperature-sensitive (ts) pleiotropic phenotype of ts A1S9 cells was investigated. Cells are ts in growth and DNA replication. They undergo decondensation of their heterochromatin, interruptions of chromatin synthesis, and changes in cell size and morphology at the non-permissive temperature (npt) of 38.5 degrees C. Whereas the rates of incorporation of 3H-leucine, 35S-methionine, and 3H-fucose into proteins were unaffected at 38.5 degrees C, net protein accumulation was greatly reduced. This imbalance resulted from a rapid increase in the rate of protein degradation at the npt. Enhancement of protein degradation was detected within 2-4 hours after temperature upshift and constitutes the earliest metabolic alteration thus far observed during expression of the temperature-sensitive phenotype. The average half-life of proteins performed in ts A1S9 cells at 34 degrees C was decreased four-fold at the npt, and all major cytoplasmic proteins were affected equally. Enhanced protein degradation at the npt was shown to be sensitive to cycloheximide, ammonia, chloroquine, and vinblastine at concentrations that did not affect the basal protein degradation of normally cycling cells. Increased protein degradation at 38.5 degrees C did not involve an equivalent increase in total cellular protease activity. The data obtained are compatible with a model that suggests that temperature inactivation of the ts A1S9 gene product results in activation of a lysosome-mediated mechanism for the rapid degradation of cytoplasmic proteins.

Hormonal Control of Deoxyribonucleic Acid and Protein Syntheses in Pea Root Cortical Explants

The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of (14)C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of (14)C-leucine into trichloroacetic acid-precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented.