Hybrid cytokines as hematopoietic growth factors.

A large body of in vitro and in vivo data suggests that combinations of cytokines provide the most effective mechanism for stimulating multilineage acceleration of hematopoiesis. Creation of a granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3) fusion protein has yielded a single therapeutic which has enhanced biological activity in comparison to the individual cytokines from which it is composed. In vivo studies with this fusion protein (PIXY321) suggest that it may provide a means to accelerate both neutrophil and platelet recovery in clinical settings in which hematopoiesis is suppressed. The biology of PIXY321 and the potential for other fusion proteins is discussed.     

Signal transduction underlying growth cone guidance by diffusible factors.

Many diffusible axon guidance cues and their receptors have been identified recently. These cues are often found to be bifunctional, acting as attractants or repellents under different circumstances. Studies of cytoplasmic signaling mechanisms have led to the notion that the response of a growth cone to a particular guidance cue depends on the internal state of the neuron, which, in turn, is under the influence of other coincident signals received by the neuron. Furthermore, many diffusible guidance cues appear to share common cytoplasmic signaling pathways.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

Molecular basis of plant growth promotion and biocontrol by rhizobacteria.

Plant-growth-promoting rhizobacteria (PGPRs) are used as inoculants for biofertilization, phytostimulation and biocontrol. The interactions of PGPRs with their biotic environment, for example with plants and microorganisms, are often complex. Substantial advances in elucidating the genetic basis of the beneficial effects of PGPRs on plants have been made, some from whole-genome sequencing projects. This progress will lead to a more efficient use of these strains and possibly to their improvement by genetic modification.     

Waste management - cytokines, growth factors and cachexia

Muscle damage with a lack of regeneration, manifests itself in several life-threatening diseases, including cancer cachexia, congestive heart failure, AIDS and sepsis. Often misdiagnosed as a condition simply of weight loss, cachexia is actually a highly complex metabolic disorder involving features of anorexia, anaemia, lipolysis and insulin resistance. A significant loss of lean body mass arises from such conditions, resulting in wasting of skeletal muscle. Unlike starvation, the weight loss seen in chronic illnesses arises equally from loss of muscle and of fat. The cachectic state is particularly problematic in cancer, typifying poor prognosis and often lowering responses to chemotherapy and radiation treatment. More than half of cancer patients suffer from cachexia, and strikingly, nearly one-third of cancer deaths are related to cachexia rather than the tumour burden. In considering this disorder, we are faced with a conundrum; how is it possible for uncontrolled growth to prevail in the tumour, in the face of unrestrained tissue loss in our muscles? Consistently, the catabolic state has been associated with a shift in the homeostatic balance between muscle synthesis and degradation mediated by the actions of growth factors and cytokines. Indeed, tumour necrosis factor-alpha (TNF-alpha) levels are raised in several animal models of cachectic muscle wasting, whereas the insulin-like growth factor (IGF) system acts potently to regulate muscle development, hypertrophy and maintenance. This concept of skeletal muscle homeostasis, often viewed as the net balance between two separate processes of protein synthesis and degradation has however changed. More recently, the view is that these two biochemical processes are not occurring independently of each other but in fact are finely co-ordinated by a web of intricate signalling networks. This review, therefore, aims to discuss data currently available regarding the mechanisms of degeneration and regeneration with specific emphasis on the potential and controversial cross-talk which may exist between anabolic growth factors (e.g. IGF-I) and catabolic cytokines (e.g. TNF-alpha). Also importantly, the potential impact at a cellular level of exercise, diet and age will be addressed. Finally, the ability to 'hi-jack' signalling pathways traditionally believed to be for growth and survival or death will be reviewed. It is anticipated that such a review will highlight significant gaps in our knowledge of the cachectic state as well as provide caution with regards to therapeutics suggesting total block on inflammatory processes such as that associated with TNF-alpha action.     

Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain.

Cloning and sequencing of cDNAs isolated from a rat cortex cDNA library reveals that a gene family encodes several highly homologous K+ channel forming (RCK) proteins. Functional characterization of the channels expressed in Xenopus laevis oocytes following microinjection of in vitro transcribed RCK-specific RNAs shows that each of the RCK proteins forms K+ channels that differ greatly in both their functional and pharmacological properties. This suggests that the molecular basis for the diversity of voltage-gated K+ channels in mammalian brain is based, at least partly, on the expression of several RCK proteins by a family of genes and their assembly to homooligomeric K+ channels with different functional properties.     

New perspectives on the design of cytokines and growth factors

A combination of molecular modelling, conventional epitope scanning and combinatorial techniques, such as phage display and DNA shuffling, has greatly improved our understanding of ligand-receptor interactions. It has therefore been possible to develop powerful cytokine-growth factor antagonists and new designer cytokines, with altered receptor specificities or with greatly enhanced biological activity. Recently, small circular peptides that mimic or block the effects of natural cytokines and growth factors have been developed; such small peptides are likely to open new avenues in therapeutics.     

Receptors and mechanisms of neurotropic effects of cytokines and growth factors

Cytokines and trophic factors (TF) are known to be involved not only into the immune processes but in majority cells, organs and physiological systems functional activity regulation as well as in pathological conditions. Cytokines and TF are shown to exert antagonistic effects on the brain, involved into local and systemic reactions modulation in response to CNS inflammation, infections and other types of injuries. Authors observed new data about cytokines and TF (particularly, the very biologically active among them - tumor necrosis factor-alpha and interleukin-1-beta) physico-chemical properties as the background for their neurotropic affects investigation. The contemporary data about cytokines and TF receptors, their interaction with neurotransmitters, penetration into the brain and their bioactivity regulation are reviewed.     

Molecular basis for Rac1 recognition by guanine nucleotide exchange factors.

Rho GTPases are activated by a family of guanine nucleotide exchange factors (GEFs) known as Dbl family proteins. The structural basis for how GEFs recognize and activate Rho GTPases is presently ill defined. Here, we utilized the crystal structure of the DH/PH domains of the Rac-specific GEF Tiam1 in complex with Rac1 to determine the structural elements of Rac1 that regulate the specificity of this interaction. We show that residues in the Rac1 beta2-beta3 region are critical for Tiam1 recognition. Additionally, we determined that a single Rac1-to-Cdc42 mutation (W56F) was sufficient to abolish Rac1 sensitivity to Tiam1 and allow recognition by the Cdc42-specific DH/PH domains of Intersectin while not impairing Rac1 downstream activities. Our findings identified unique GEF specificity determinants in Rac1 and provide important insights into the mechanism of DH/PH selection of GTPase targets.     

Autocrine and paracrine regulation by cytokines and growth factors in melanoma

Tumour development and progression involves the expression of oncogenes and inactivation of tumour suppressor genes, leading to the appearance of multiple malignant characteristics. Malignant melanoma cells express different growth factors and cytokines and their receptors in respective stages of tumour progression, which by autocrine and paracrine effects enable them to grow autonomously and confer competence to metastasis. Autocrine growth factors (bFGF, MGSA/GRO, IL-8 and sometimes IL-6, PDGF-A, IL-10) produced by melanoma cells stimulate proliferation of the producing cell itself, while paracrine growth factors (for example PDGF, EGF, TGF-beta, IL-1, GM-CSF, IGF-I, NGF, VEGF) modulate the microenvironment to the benefit of tumour growth and invasion. Paracrine effects include angiogenesis, stroma formation, modulation of host immune response, activation of proteolytic enzymes, adhesion or motility and metastasis formation. Some growth factors have inhibitory effects on melanocytes and early lesions (IL-1, IL-6, TGF-beta, OSM, TNF and IFN) but not on advanced stage melanomas, and in some cases they switch to autocrine stimulator (IL-6, TGF-beta). Understanding the involvement of different growth factors and cytokines in the molecular mechanism of melanoma progression will help to provide an insight into new future therapeutic approaches for melanoma.     

Molecular genetic defect underlying alpha-L-iduronidase pseudodeficiency.

Mucopolysaccharidosis type I (i.e., Hurler, Hurler-Scheie, and Scheie syndromes) and type II (i.e., Hunter syndrome) are lysosomal storage disorders resulting from alpha-L-iduronidase (IDUA) deficiency and iduronate-2-sulfatase (IDS) deficiency, respectively. The a priori probability that both disorders would occur in a single individual is approximately 1 in 5 billion. Nevertheless, such a proband was referred for whom clinical findings (i.e., a male with characteristic facies, dysostosis multiplex, and mental retardation) and biochemical tests indicated these concomitant diagnoses. In repeated studies, leukocyte 4 methylumbelliferyl-alpha-L-iduronidase activities in this kindred were as follows: <1.0 nmol/mg protein/h in the proband and proband's clinically normal sister; 45.3 in mother; and 45.7 in father (normal range 65.0-140). Leukocyte L-O-(alpha-iduronate-2-sulfate)-(1->4)-D-O-2,5-anhydro[1-3H]mannitol-6- sulfate activities were as follows: 0.0 U/mg protein/h in the proband; 5.7 in his sister; 4.9 in mother; and 15.0 in father (normal range 11.0-18.4). Multiple techniques, including automated sequencing of the entire IDS and IDUA coding regions, were employed to unravel the molecular genetic basis of these intriguing observations. The common IDS mutation R468W was identified in the proband, his mother, and his sister, thus explaining their biochemical phenotypes. Additionally, the proband, his sister, and his father were found to be heterozygous for a common IDUA mutation, W402X. Notably, a new IDUA mutation A300T was also identified in the proband, his sister, and his mother, accounting for reduced IDUA activity in these individuals; the asymptomatic sister, whose cells demonstrated normal glycosaminoglycan metabolism, is thus a compound heterozygote for W402X and the new allele. This A300T mutation is the first IDUA pseudodeficiency gene to be elucidated at the molecular level.     

Molecular basis of symbiotic promiscuity.

Eukaryotes often form symbioses with microorganisms. Among these, associations between plants and nitrogen-fixing bacteria are responsible for the nitrogen input into various ecological niches. Plants of many different families have evolved the capacity to develop root or stem nodules with diverse genera of soil bacteria. Of these, symbioses between legumes and rhizobia (Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium) are the most important from an agricultural perspective. Nitrogen-fixing nodules arise when symbiotic rhizobia penetrate their hosts in a strictly controlled and coordinated manner. Molecular codes are exchanged between the symbionts in the rhizosphere to select compatible rhizobia from pathogens. Entry into the plant is restricted to bacteria that have the "keys" to a succession of legume "doors". Some symbionts intimately associate with many different partners (and are thus promiscuous), while others are more selective and have a narrow host range. For historical reasons, narrow host range has been more intensively investigated than promiscuity. In our view, this has given a false impression of specificity in legume-Rhizobium associations. Rather, we suggest that restricted host ranges are limited to specific niches and represent specialization of widespread and more ancestral promiscuous symbioses. Here we analyze the molecular mechanisms governing symbiotic promiscuity in rhizobia and show that it is controlled by a number of molecular keys.     

Molecular basis of insulin resistance.

The recent application of recombinant DNA technology to clinical investigation now allows the identification of the molecular alterations responsible for insulin resistance. In this review, the recent knowledge concerning these investigations is reported. Genetic mutations of the insulin gene as the source of insulin resistance have been reported for a long time. More recently a series of mutations of the insulin receptor gene have been identified as the cause of the extreme insulin resistance, observed in rare syndromes, such as type A insulin resistance or leprechaunism. However, it is probable that the majority of the molecular defects causing insulin resistance occur at the postreceptor level. The key proteins involved in the different intracellular signalling pathways of insulin are only partly identified. A better understanding of the mechanisms of insulin action is essential for the identification of corresponding genetic alterations. The investigations concerning the glucose transporter GLUT4 and glucokinase genes are good examples of complex but promising research, which has recently started. Elucidation of the genetic and molecular basis of diseases such as type II diabetes or other states associated with insulin resistance, is the long-term goal.