"Whey" proteins of milk of the red (Macropus rufus) and eastern grey (Macropus giganteus) kangaroo

1. A comparison is made of gel electrophoretic patterns of the "whey" proteins of the milk of red (Macropus rufus) and eastern grey (Macropus giganteus) kangaroos at various stages of lactation. Qualitative and quantitative changes occur with time during the mature phase of lactation of both types. Their onset is related solely to the stage of lactation. "Whey" proteins are isolated and characterised and the nature of protein changes determined for the first time. 2. The anodic electrophoretic pattern is divided into 6 main zones (designated A F in order of decreasing mobility) and 2 cathodic zones (G and H) that are only detected in the milk of M. giganteus. 3. Zones A, B and C are milk specific. Zone B is present throughout lactation in both species and is an alpha-lactalbumin. Zones A and C are present only in late lactation, zone C, usually, but not always, appearing first. Zone A is an alpha-lactalbumin in M. giganteus, but is not an alpha-lactalbumin in M. rufus. Zone C appears to be the same protein in both species and is possibly a beta-lactoglobulin. 4. Zone D is kangaroo serum albumin and zone E is possibly a beta 2-microglobulin. Zone F contains three main iron (III) binding bands whose relative intensity varies with stage of lactation. Their intensity differs from the corresponding blood serum transferrin bands. 5. Zone H of Macropus giganteus is a lysozyme. 6. Lactose is present in the milk, but is not the principal sugar. 7. The significance of the results is discussed.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

The Discovery of a Reciprocal Relationship between Tyrosine-Kinase Signaling and Cullin Neddylation

While neddylation is known to activate cullin (CUL)-RING ubiquitin ligases (CRLs), its role in regulating T cell signaling is poorly understood. Using the investigational NEDD8 activating enzyme (NAE) inhibitor, MLN4924, we found that neddylation negatively regulates T cell receptor (TCR) signaling, as its inhibition increases IL-2 production, T cell proliferation and Treg development in vitro. We also discovered that loss of CUL neddylation occurs upon TCR signaling, and CRLs negatively regulate IL-2 production. Additionally, we found that tyrosine kinase signaling leads to CUL deneddylation in multiple cell types. These studies indicate that CUL neddylation is a global regulatory mechanism for tyrosine kinase signaling.     

Prevention of experimental autoimmune encephalomyelitis in common marmosets using an anti-IL-12p40 monoclonal antibody

The experimental autoimmune encephalomyelitis (EAE) model in the common marmoset approximates recognized features of the human disease multiple sclerosis (MS) with regard to its clinical presentation as well as neuropathological and radiological aspects of the lesions in brain and spinal cord. IL-12 is a proinflammatory cytokine that is produced by APC and promotes differentiation of Th1 effector cells. IL-12 is produced in the developing lesions of patients with MS as well as in EAE-affected animals. Previously it was shown that interference in IL-12 pathways effectively prevents EAE in rodents. In this study we report that in vivo neutralization of IL-12p40 using a novel Ab has beneficial effects in the myelin-induced EAE model in common marmosets. The Ab was injected i.v. at 7-day intervals starting well after immunization (day 14) and was continued until the end of the study (day 86). Stable levels of the Ab were measured 3 days after each injection throughout the study period. During this period anti-Ab responses could not be detected. We demonstrate that anti-IL-12p40 treatment has a protective effect on the neurological dysfunction as well as on neuropathological changes normally observed in the brain and spinal cord of EAE-affected individuals.     

Anti-TNF-alpha antibody allows healing of joint damage in polyarthritic transgenic mice

Anti-tumor-necrosis-factor-alpha (TNF-alpha) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-alpha (hTNF-alpha) and develop a progressive polyarthritic disease. Treatment of both young (7- or 8-week-old) and aged (27- or 28-week-old) mice commenced when at least two limbs showed signs of moderate to severe arthritis. The therapeutic efficacy of anti-TNF-alpha antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-alpha treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-alpha prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-alpha and local expression of various proinflammatory mediators were all diminished by anti-TNF-alpha treatment, confirming a critical role of hTNF-alpha in this model of progressive polyarthritis. More importantly, the amelioration of the disease was associated with reversal of existing structural damage, including synovitis and periosteal bone erosions evident on histology. Repair of cartilage was age dependent: reversal of cartilage degradation after anti-TNF-alpha treatment was observed in young mice but not in aged mice.