After the completion of the human and other genome projects it emerged that the number of genes in organisms as diverse as fruit flies, nematodes, and humans does not reflect our perception of their relative complexity. Here, we provide reliable evidence that the size of protein interaction networks in different organisms appears to correlate much better with their apparent biological complexity. We develop a stable and powerful, yet simple, statistical procedure to estimate the size of the whole network from subnet data. This approach is then applied to a range of eukaryotic organisms for which extensive protein interaction data have been collected and we estimate the number of interactions in humans to be Ϸ650,000. We find that the human interaction network is one order of magnitude bigger than the Drosophila melanogaster interactome and Ϸ3 times bigger than in Caenorhabditis elegans.evolutionary systems biology ͉ network inference ͉ network sampling theory ͉ network evolution O ne of the perhaps most surprising results of the genomesequencing projects was that the number of genes is much lower than had been expected and is, in fact, surprisingly similar for very different organisms (1, 2). For example, the nematode Caenorhabditis elegans appears to have a similar number of genes as humans, whereas rice and maize appear to have even more genes than humans. It was then quickly suggested that the biological complexity of organisms is not reflected merely by the number of genes but by the number of physiologically relevant interactions (1, 3). In addition to alternative splice variants (4), posttranslational processes (5), and other (e.g., genetic) factors influencing gene expression (6, 7), the structure of interactome is one of the crucial factors underlying the complexity of biological organisms. Here, we focus on the wealth of available protein interaction data and demonstrate that it is possible to arrive at a reliable statistical estimate for the size of these interaction networks. This approach is then used to assess the complexity of protein interaction networks in different organisms from present incomplete and noisy protein interaction datasets.There are now fairly extensive protein interaction network (PIN) datasets in a number of species, including humans (8, 9). These have been generated by a variety of experimental techniques (as well as some in silico inferences). Although these techniques and the resulting data are (i) notoriously prone to false positives and negatives (10, 11), and (ii) result in highly idealized and averaged network structures (12), such interaction datasets are increasingly turning into useful tools for the analysis of the functional (e.g., ref. 13) and evolutionary properties (14) of biological systems. In particular, in Saccharomyces cerevisiae we are beginning to have a fairly complete description of the protein interaction network that is accessible with current experimental technologies; the recent high-quality literaturecurated dataset of Reguly et al. (15) provides us w...

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like zymogen that is activated toTAFIa by plasmin, thrombin, or the thrombin-thrombomodulin complex. The enzyme TAFIa attenuates clot lysis by removing lysine residues from a fibrin clot. Screening of nine human cDNA libraries indicated a common variation in TAFI at position 325 (Ile-325 or Thr-325). This is in addition to the variation at amino acid position 147 (Ala-147 or Thr-147) characterized previously. Thus, four variants of TAFI having either Ala or Thr at position 147 and either Thr or Ile at position 325 were stably expressed in baby hamster kidney cells and purified to homogeneity. The kinetics of activation of TAFI by thrombin/thrombomodulin were identical for all four variants; however, Ile at position 325 extended the half-life of TAFIa from 8 to 15 min at 37°C, regardless of the residue at position 147. In clot lysis assays with thrombomodulin and the TAFI variants, or with pre-activated TAFI variants, the Ile-325 variants exhibited an antifibrinolytic effect that was 60% greater than the Thr-325 variants. Similarly, in the absence of thrombomodulin, the Ile-325 variants exhibited an antifibrinolytic effect that was 30 -50% greater than the Thr-325 variants. In contrast, the variation at position 147 had little if any effect on the antifibrinolytic potential of TAFIa. The increased antifibrinolytic potential of the Ile-325-containing TAFI variants reflects the fact that these variants have an increased ability to mediate the release of lysine from partially degraded fibrin and suppress plasminogen activation. These findings imply that individuals homozygous for the Ile-325 variant of TAFI would likely have a longer lived and more potent TAFIa enzyme than those homozygous for the Thr-325 variant. Thrombin-activable fibrinolysis inhibitor (TAFI)1 is a zymogen found in human plasma (1), which is also known as plasma procarboxypeptidase B (2) and procarboxypeptidase U (3). It can be activated by thrombin (1), plasmin (4), or the thrombinthrombomodulin complex (5) to the carboxypeptidase B-like enzyme, TAFIa. When exposed to a fibrin clot, TAFIa catalyzes the removal of carboxyl-terminal lysines, thereby diminishing the cofactor activity for plasminogen activation (6). Less efficient plasminogen activation on the fibrin clot corresponds to prolongation of fibrinolysis, and in this way TAFIa can serve as a potent antifibrinolytic enzyme. Studies performed using an in vitro human plasma model have found that clot lysis times can be attenuated up to 3-fold in the presence of TAFIa as compared with clots lysed in the absence of TAFIa (5).Activation of TAFI is catalyzed only slowly by thrombin alone; however, in the presence of thrombin/thrombomodulin, the efficiency of activation is increased 1000-fold. Despite the large thrombomodulin dependence of TAFI activation, in vitro clot lysis assays done in the absence of thrombomodulin still exhibit prolonged clot lysis times as compared with similar assays performed with TAFI-depleted plas...

Because of turnover, protein synthesis and breakdown can each be involved in the regulation of the growth of tissue protein. To investigate the regulation of skeletal-muscle-protein growth we measured rates of protein synthesis and breakdown in growing rats during development on a good diet, during development on a marginally low-protein diet and during rehabilitation on a good diet after a period of severe protein deficiency. Rates of protein synthesis were measured in vivo with a constant intravenous infusion of [14C]tyrosine. The growth rate of muscle protein was measured and the rate of breakdown calculated as breakdown rate=synthesis rate-growth rate. These measurements showed that during development on a good diet there was a fall with age in the rate of protein synthesis resulting from a fall in capacity (RNA concentration) and activity (synthesis rate per unit of RNA). There was a fall with age in the breakdown rate so that the rate was highest in the weaning rats, with a half-life of 3 days. There was a direct correlation between the fractional growth and breakdown rates. During rehabilitation on the good diet, rapid growth was also accompanied by high rates of protein breakdown. During growth on the inadequate diet protein synthesis rates were lesss than in controls, but growth occurred because of decreased rates of protein breakdown. This compression was not complete, however, since ultimate muscle size was only one-half that of controls. It is suggested that increased rates of protein breakdown are a necessary accompaniment to muscle growth and may result from the way in which myofibrils proliferate.

Vampire bat plasminogen activator (b-PA) causes less fibrinogen (Fg) consumption than tissue-type plasminogen activator (t-PA). Herein, we demonstrate that this occurs because the complex of D-dimer noncovalently linked to fragment E ((DD)E), the most abundant degradation product of cross-linked fibrin, as well as Fg, stimulate plasminogen (Pg) activation by t-PA more than b-PA. To explain these findings, we characterized the interactions of t-PA, b-PA, Lys-Pg, and Glu-Pg with Fg and (DD)E using right angle light scattering spectroscopy. In addition, interactions with fibrin were determined by clotting Fg in the presence of various amounts of t-PA, b-PA, Lys-Pg, or Glu-Pg and quantifying unbound material in the supernatant after centrifugation. Glu-Pg and Tissue-type plasminogen activator (t-PA)1 is a naturally occurring serine protease that initiates fibrinolysis by converting plasminogen (Pg) to plasmin (1). Not only is fibrin the target for plasmin attack, but fibrin also stimulates t-PA-mediated Pg activation (2, 3). To accomplish this, fibrin acts as a template to which both t-PA and Pg bind (4). The fibrin-binding properties of t-PA have been ascribed to its finger and second kringle (K 2 ) domains (5, 6), although recent studies suggest that the protease domain also influences the interaction of t-PA with fibrin (4, 7, 8). The binding of both Glu-and Lys-plasminogen (Glu-Pg and Lys-Pg, respectively) to fibrin is entirely kringle-mediated, with Lys-Pg having higher affinity for fibrin than Glu-Pg (9). As a functional consequence of t-PA interaction with fibrin, the catalytic efficiency of t-PA-mediated Pg activation is 2-3 orders in magnitude higher in the presence of fibrin than in its absence (3, 10). In contrast to fibrin, fibrinogen (Fg) stimulates Pg activation by t-PA only 25-fold (3, 10). Based on these considerations, t-PA is designated a fibrin-specific plasminogen activator (11). Despite this designation, t-PA causes systemic plasminemia and fibrinogenolysis when given to patients (12). In recent studies, we have demonstrated that t-PA causes systemic plasminemia, because, like intact fibrin, soluble fibrin degradation products stimulate t-PA-mediated Pg activation (13). Furthermore, we have identified the (DD)E complex as the fibrin derivative primarily responsible for this effect (14) and have shown that the stimulatory activity of (DD)E is similar to that of fibrin.2 (DD)E, a complex of D-dimer noncovalently bound to fragment E, is the major degradation product of cross-linked fibrin (15). As a potent stimulator of t-PA-mediated activation of Pg, (DD)E generated during thrombus dissolution has the potential to induce systemic plasminemia (12,15).The limited fibrin specificity of t-PA has prompted the development of plasminogen activators with greater selectivity for fibrin (16). One such agent is the plasminogen activator isolated from the saliva of vampire bats (Desmodus rotundus) (17). Full-length vampire bat salivary plasminogen activator (designated DSPA␣ 1 ) has over 72% amino acid seque...

I. Colonies of rats have been maintained for twelve generations on diets adequate (dietaryprotein energy: total metabolizable energy (NDP :E) 0.1) or marginally deficient in protein (NDP: E 0.068).2. In the malnourished colony, the proportion of 'small-for-gestational-age' offspring was ten times as high as amongst the well-nourished colony, growth was slow, sexual maturation delayed, especially in the females, and, when adult, both sexes were significantly lighter and shorter than adults of the well-nourished colony. Organs, other than the eye, weighed less than those of well-nourished 'age' controls, but when expressed relative to body-weight, the brain, pituitary, thyroid, adrenals, testes, thymus and eyes were larger, the pancreas unchanged and the kidneys smaller than those of the well-nourished 'age' controls. The relative weight of the liver showed little change, being slightly increased in the males and, like the ovaries, slightly reduced in the females. On a body-weight basis, the brains were about 50 yo heavier than normal, but in absolute terms were 5 3 . 5 % lighter than those of the wellnourished animals, the cerebellum (10.5 % lighter in males and 12.9 % lighter in females) being more severely affected than the cerebrum (4 yo lighter). 3.The young malnourished rats showed increased exploratory activity, transient head tremors and an increased sensitivity to noise, the latter being long-lasting if not permanent. When adult, they showed marked differences in behaviour and learning patterns and it was difficult to attract and hold their attention. In situations demanding a choice the animals were very excited, emitted loud squeals and tried to escape from what was clearly a stressful situation. However, a casual examination of the malnourished adults revealed a rather small, badly groomed, excitable rat without gross abnormalities.4. The findings are discussed in relation to changes found in malnourished human communities.There are many reports (see review by Platt & Stewart, 1971) indicating that protein-energy deficiency (PED) affects growth, development and reproduction as well as the chemical composition and morphology of many organs. Much effort has been expended on investigations into the effects of PED on brain development. The timing, duration and severity of the imposed deficiencies have varied greatly. Mothers have been maintained on inadequate supplies of food during gestation, the young have been given only limited access to the mother, the number of young suckled by one dam has been increased, animals have been given deficient diets only from weaning, and in some instances investigators have combined two or more of these techniques. Under any of these conditions the diet has to be severely restricted in quantity or quality, or both, in order to establish a deficiency during the critical periods of development. Except during periods of war or famine these are not the conditions existing in human communities in the depressed or non-industrialized areas of the world. More commonly the people...