Variations In Protein Synthesis Research Articles

A preliminary study on growth and protein synthesis of juvenile barramundi, Lates calcarifer at different temperatures

Temperature is recognized to be the most important environmental factor affecting growth and protein synthesis in fish. The optimal temperature for growth of juvenile barramundi is 31 °C, although culture often occurs at temperatures which are above and below this optimum. Juveniles (2.96 ± 0.46 g) were held at five different temperatures ranging from 21 to 33 °C at 3 °C intervals. Fish were fed to satiation twice daily (504.5 g kg − 1 crude protein, 190.5 g kg − 1 lipid, 128.5 g kg − 1 ash, and 20.2 GE MJ kg − 1 ). Daily feed intake (g), growth (% d − 1 ), growth efficiency, and protein synthesis (measured 24 h after feeding) were determined for each temperature. Feed intake was significantly higher at 33 °C, than at any other temperature. Growth and growth efficiency were not significantly different between the 27, 30 and 33 °C groups but were significantly higher than the 21 and 24 °C groups. In order to take account of the variation in protein synthesis over the 24 h following feeding and model daily protein turnover, daily rates of protein synthesis were estimated from previously determined relationships between white muscle and whole body rates of protein synthesis. This showed that protein synthesis was not significantly different between 27 and 33 °C, synthesis retention efficiency was over 40% at these temperatures, and at 21 °C growth efficiency was poor. Growth efficiency and protein metabolism were optimal over a temperature from 27 to 33 °C.

Molecular Profiling of Angiogenic Markers: A Step Towards Interpretive Analysis of a Complex Biological Function

Gene expression profiling is now being used routinely to define complex biological events. The profiling of a large array of genes expressed in the progression of a biological response opens the door to our understanding the unique relationships between genes and their functions. Angiogenesis, the process of new blood vessel development, is a necessary component of both normal and pathological physiology. In this issue of The American Journal of Pathology, Shih and colleagues 1 have used quantitative molecular profiling of angiogenic-related factors to define some of the elements required for angiogenic profiling. Although presented as a technical advance, the basic concept of this work is that the use of quantitative molecular profiling of gene expression gives additional insight into functional interrelationships between the genes expressed during the angiogenic process. This approach can be applied in large array format as a diagnostic tool for experimental systems and pathological samples.

In vivo longitudinal variations in protein synthesis in developing ovine intestines.

Changes in fractional rates of protein synthesis (Ks) were investigated at different small and large intestinal sites in 1-, 5-, and 8-wk-old milk-fed and 8-wk-old weaned lambs, a species with early intestinal maturation similar to most domestic animals and humans, with the use of a flooding dose of L-[3H]valine. Between 1 and 8 wk of age, Ks did not change significantly in the duodenum, the cecum, or the colon of milk-fed lambs, but was depressed by 30% in the jejunum and by 39% in the ileum. This was because of reduced ribosomal capacity, i.e., total RNA-to-protein ratio (Cs) in the jejunum, and also alterations in both Cs and protein synthetic efficiency, i.e., rate of synthesis relative to RNA (KRNA) in the ileum. Ks values throughout the small intestine were significantly higher (45-55%) in weaned lambs than in 8-wk-old milk-fed animals. This enhancement of protein synthesis was mainly related to an increase in KRNA (27-40%). Ks decreased by 43% from the duodenum to the ileum in both milk-fed and weaned 8-wk-old animals, but not in 1- and 5-wk-old milk-fed lambs, because of a marked reduction in KRNA. It was concluded that changes in nutrients at weaning, weaning itself, or both, enhanced protein synthesis without any specific effect on small intestinal site. By contrast, intrinsic developmental factors were responsible only for the regional differences in small intestinal Ks that occurred at 8 wk of age. Longitudinal variations in protein synthesis may contribute to the establishment of the well-recognized jejunoileal gradients of brush-border enzymes and villus height that characterize the mature mammalian small intestine.

Protein Synthesis during Lactation: No Circadian Variation in Mammary Gland and Liver of Rats Fed Diets Varying in Protein Quality and Level of Intake

Circadian changes in protein synthesis were studied in lactating rats fed diets varying in protein quality and feeding level. At parturition, rats previously fed a stock diet were assigned to semipurified diets suppyling 23% protein. Two groups were fed wheat gluten (WG) or casein (C) ad libitum; a third group was pair-fed casein to consumption of the WG group (C-PF). At four different times on day 15 of lactation, protein synthesis was measured in vivo in mammary gland, liver and calf muscle with a large dose of L-[4-3H]phenylalanine. Pup weights at day 15 for dams fed WG, C and C-PF were 19.3 ± 0.3, 33.9 ± 0.5 and 27.2 ± 0.5 g, respectively. No significant circadian variation in fractional synthesis rate (FSR) was observed in either mammary gland or liver. Higher variability in muscle FSRs precludes conclusions in regard to this tissue. However, protein quality and level of feeding both affected FSR. For rats fed WG, C and C-PF, FSRs were, respectively: 77 ± 4, 116 ± 6 and 64 ± 3%/day in mammary gland; 67 ± 4, 78 ± 5 and 74 ± 4%/day in liver; and 3.4 ± 0.5, 6.7 ± 1.0 and 4.0 ± 0.8%/day in muscle. Absolute synthesis rates were, respectively: 584 ± 60, 1743 ± 148 and 663 ± 58 mg/day in mammary gland; and 469 ± 46, 970 ± 72 and 702 ± 62 mg/day in liver. The results confirm that dietary protein quality and feeding level affect protein synthesis rates in lactating rats and demonstrate that there is no circadian variation in protein synthesis in mammary gland and liver of lactating rats.

Role of attenuation in growth rate-dependent regulation of the S10 r-protein operon of E. coli.

We have investigated the transcription of the 11 gene S10 ribosomal protein operon of Escherichia coli under various growth conditions. The differential synthesis rate of structural gene message increases 2- to 2.5-fold immediately after a shift-up from glycerol minimal medium to glucose plus amino acids. After the initial increase, the transcription rate goes through several oscillations before reaching the new steady-state rate. By comparing the rates of transcription of leader and structural genes, we conclude that these oscillations are due predominantly to changes in the level of read-through at the S10 attenuator. This regulation of attenuation can account for most of the variations in protein synthesis from the S10 operon after a shift. We also measured the level of read-through in cells growing exponentially in different growth media. Over a 2.5-fold range in growth rates, the read-through changed less than 50%. Thus, regulation of attenuation cannot explain the growth-dependent regulation of ribosomal protein synthesis during steady-state growth. Apparently, additional mechanisms are required to control the expression of the S10 operon in exponentially growing cells.

Variations in Protein Synthesis in Different Regions of Greening Leaves of Barley Seedlings and Effects of Imposed Water Stress

Water stress, applied to the roots of six-day-old barley seedlings markedly reduced the capacity of their leaves to synthesize proteins during greening, a process that normally involves intense synthetic activity. Upon illumination of etiolated seedlings protein synthesis commenced most rapidly in the basal region, and then declined in activity. Thereafter, in turn, the middle and apical regions exhibited a similar pattern of synthetic activity; this is indicative of a ‘wave’ of protein synthesis progressing from the base of the leaf to the apex as greening proceeded. The synthesis of proteins varied both quantitatively and qualitatively in different regions of the leaf during greening. For example, there was a noticeable increase in ribulose-1, 5-bisphosphate carboxylase synthesis in the apical region of the leaf compared to the basal region. Water stress reduced protein synthesis in all regions of the leaf, although most effectively in the oldest, apical regions. Upon return to full water status, the basal regions recovered the most rapidly and to the greatest extent. Similar results were obtained when both intact greening leaves, and isolated segments from different regions of the leaf were used. The reduction in protein synthesis elicited by water stress was not due to a selective quantitative change in any particular protein; one protein, of an approximate molecular weight of 60 kD, appeared to be synthesized only under stress conditions.

Role of non-histones in chromosome structure. Cell cycle variations in protein synthesis.

As part of a study of the role of non-histone proteins in chromosome structure, the synthesis of non-histones associated with interphase chromatin was investigated. Synchronized suspension cultures of HeLa cells were pulse-labeled with [35S]methionine, and chromatin was prepared by mild micrococcal nuclease digestion. Two-dimensional polyacrylamide gel electrophoresis, in addition to one-dimensional electrophoresis, was used to resolve the patterns of incorporation of radioactive label. Significant variations in non-histone synthesis were seen during the cell cycle. A strong correlation was not found between DNA synthesis in mid-S phase and variations in non-histone synthesis. The non-histone proteins of purified metaphase chromosomes were also characterized by two-dimensional gel electrophoresis and compared to the proteins of interphase chromatin. The pattern of non-histones is not identical with that of interphase chromatin, although a number of major species may be shared by interphase chromatin and metaphase chromosomes. The HeLa nuclear scaffold, the framework that maintains the overall morphology of the interphase nucleus, shows relatively few proteins on two-dimensional gels. The synthesis of nuclear scaffold proteins was quantitated by excising each of 19 proteins from two-dimensional gels and determining the incorporated radioactivity by scintillation counting. Substantial variations in protein synthesis were found, with several species showing changes of about 2-fold in the percentage of incorporation.

Carbohydrate dependent protein synthesis and enzyme activity in the jejunum

The mechanism by which dietary carbohydrate (CH) regulates jejunal disaccharidase activity has not been elucidated. Studies were undertaken to determine whether adaptive changes in disaccharidase levels were accompanied by variations in protein synthesis; and if so, whether they were related to cell migration and RNA synthesis. 200 g rats were fed a CH-free diet (with isocaloric protein replacement) for 7 days and then fed a diet rich in either sucrose (68% cal) or starch (51% cal). Incorporation of 14C amino acids into total mucosal and brush border (BB) proteins (TCA insoluble) was measured. Both sucrose and starch stimulated an increase of 30% in the labeling of total mucosal proteins and greater than 100% rise in that of BB proteins. Simultaneously, there was 100% rise in sucrose and lactase activity and 60% increase in maltase activity in crude jejunal homogenate and BB. When actinomycin-D was injected IP 6 hrs prior to feeding sucrose, RNA synthesis measured 24 hrs later was reduced 50%, and cell migration was arrested. Although neither basal rates of total mucosal protein synthesis nor the increase stimulated by CH feeding were inhibited, 14C labelling of the BB was reduced. Sucrase, lactase and maltase levels in the crude homogenates rose 75–100%, but these activities in the BB did not increase in actinomycin-D treated animals. The data suggest that dietary CH controls disaccharidase acitivity by changes in protein synthesis independent of RNA synthesis and cell migration, and support the concept that there is an RNA-dependent step required for the transfer of newly formed disaccharidases from sites of synthesis to the BB.

Cell-free cyclic variation of protein synthesis associated with the cell cycle in sea urchin embryos.

Cyclic variation in protein synthesis was studied in cell-free systems from sea urchin eggs. The periodicity of the cycle observed in the 12,000 × g supernatant of fertilized eggs was inversely proportional to the concentration of protein in the incubation medium over a certain concentration range and was not affected by temperature under the experimental conditions. Addition of the 12,000×g supernatant from fertilized eggs induced cyclic variation in the 12,000 ×g supernatant from unfertilized eggs. The active factors inducing the cyclic variation were present in the postmicrosomal fraction, not in the ribosomes. Two macromolecular factors and a class of low molecular weight factors in the postmicrosomal fraction were found to be necessary for induction of cyclic variation. One of the macromolecular factor was identified as maternal messenger RNA. The low molecular weight factor was heat stable and seemed to be composed of more than one component. It could be partially replaced by a mixture of ATP and GTP. The maternal messenger RNA and the low molecular weight factor were not present in an active state in the supernatant of unfertilized eggs, but appeared in the supernatant very soon after fertilization. A fraction precipitated at 12,000×g was necessary for their appearance. Another factor, which may control the phase and amplitude of the cyclic variation, was found in the macromolecular fraction of supernatant of both fertilized and unfertilized eggs. Experiments on induction of cyclic variation in the supernatant of unfertilized eggs by addition of these factors suggested that maternal messenger RNA and the low molecular weight factor act as developers of activity for protein synthesis, while another macromolecular factor acts as a pace-maker. Regular latent fluctuation of this factor is assumed to occur leading to cyclic variation of synthesis. Cyclic variation was initiated by elevation of the activity in the supernatant of eggs on fertilization or of unfertilized eggs on homogenization. The cycle initiated in intact cells by fertilization continued in the supernatant of these cells after homogenization in a modified way. The relationship between the cell-free cycle and the cycle in intact cells is discussed.

Cytoplasmic regulation and cyclic variation in protein synthesis in the early cleavage stage of the sea urchin embryo

Protein synthesis in intact sea urchin embryos in the early cleavage stage showed cyclic variation superimposed on an increasing basal rate of synthesis. The cyclic variations in protein synthesis seem to be correlated with mitotic division, and may be directly related to the mitotic cycle. The length of the cycles in intact embryos was inversely correlated with the temperature, but the amount of protein synthesized during each cycle was approximately the same regardless of the temperature. Cyclic fluctuation in protein synthesis, initiated by fertilization, was observed even in the presence of actinomycin D. Cycles of protein synthesis were observed even in the absence of nuclear and cytoplasmic division due to treatment with colchicine, puromycin, and cycloheximide. Similar cyclic variation, but in mirror image to that of protein synthesis, was observed in proteolytic activity. Cell-free systems (12,000 g supernatant), where practically no protease activity was found, of sea urchin embryos at various stages were shown to carry out protein synthesis at rates varying in a cyclic fashion without increase in the basal level of synthesis. These cyclic changes were not observed in intact unfertilized eggs or in cell-free systems prepared from them. The periodic appearance and disappearance of polyribosomes corresponding to the cell-free cycle was observed on incubation of the 12,000 g supernatant from fertilized eggs. It was suggested that the cytoplasm may regulate protein synthesis, resulting ultimately in mitotic division. Continuous and discontinuous protein synthesis in this stage and the nature of cyclic protein synthesis are discussed.