Antibodies to ribosomal P proteins of Trypanosoma cruzi in Chagas disease possess functional autoreactivity with heart tissue and differ from anti-P autoantibodies in lupus

Anti-P antibodies present in sera from patients with chronic Chagas heart disease (cChHD) recognize peptide R13, EEEDDDMGFGLFD, which encompasses the C-terminal region of the Trypanosoma cruzi ribosomal P1 and P2 proteins. This peptide shares homology with the C-terminal region (peptide H13 EESDDDMGFGLFD) of the human ribosomal P proteins, which is in turn the target of anti-P autoantibodies in systemic lupus erythematosus (SLE), and with the acidic epitope, AESDE, of the second extracellular loop of the β1-adrenergic receptor. Anti-P antibodies from chagasic patients showed a marked preference for recombinant parasite ribosomal P proteins and peptides, whereas anti-P autoantibodies from SLE reacted with human and parasite ribosomal P proteins and peptides to the same extent. A semi-quantitative estimation of the binding of cChHD anti-P antibodies to R13 and H13 using biosensor technology indicated that the average affinity constant was about 5 times higher for R13 than for H13. Competitive enzyme immunoassays demonstrated that cChHD anti-P antibodies bind to the acidic portions of peptide H13, as well as to peptide H26R, encompassing the second extracellular loop of the β1 adrenoreceptor. Anti-P antibodies isolated from cChHD patients exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats, which resembles closely that of anti-β1 receptor antibodies isolated from the same patient. In contrast, SLE anti-P autoantibodies have no functional effect. Our results suggest that the adrenergic-stimulating activity of anti-P antibodies may be implicated in the induction of functional myocardial impairments observed in cChHD.

Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development.

Factors that regulate cellular migration during embryonic development are essential for tissue and organ morphogenesis. Scatter factor/hepatocyte growth factor (SF/HGF) can stimulate motogenic and morphogenetic activities in cultured epithelial cells expressing the Met tyrosine kinase receptor and is essential for development; however, the precise physiological role of SF/HGF is incompletely understood. Here we provide functional evidence that inappropriate expression of SF/HGF in transgenic mice influences the development of two distinct migratory cell lineages, resulting in ectopic skeletal muscle formation and melanosis in the central nervous system, and patterned hyperpigmentation of the skin. Committed TRP-2 positive melanoblasts were found to be situated aberrantly within defined regions of the transgenic embryo, including the neural tube, which overproduced SF/RGF. Our data strongly suggest that SF/HGF possesses physiologically relevant scatter activity, and functions as a true morphogenetic factor by regulating migration and/or differentiation of select populations of premyogenic and neural crest cells during normal mammalian embryogenesis.

Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes.

A mutation within the obese gene was recently identified as the genetic basis for obesity in the ob/ob mouse. The obese gene product, leptin, is a 16-kDa protein expressed predominantly in adipose tissue. Consistent with leptin's postulated role as an extracellular signaling protein, human embryonic kidney 293 cells transfected with the obese gene secreted leptin with minimal intracellular accumulation. Upon differentiation of 3T3-L1 preadipocytes into adipocytes, the leptin mRNA was expressed concomitant with mRNAs encoding adipocyte marker proteins. A factor(s) present in calf serum markedly activated expression of leptin by fully differentiated 3T3-L1 adipocytes. A 16-hr fast decreased (by approximately 85%) the leptin mRNA level of adipose tissue of lean (ob/+ or +/+) mice but had no effect on the approximately 4-fold higher level in obese (ob/ob) littermates. Since the mutation at the ob locus fails to produce the functional protein, yet its cognate mRNA is overproduced, it appears that leptin is necessary for its own downregulation. Leptin mRNA was also suppressed in adipose tissue of rats during a 16-hr fast and was rapidly induced during a 4-hr refeeding period. Insulin deficiency provoked by streptozotocin also markedly down-regulated leptin mRNA and this suppression was rapidly reversed by insulin. These results suggest that insulin may regulate the expression of leptin.

A mouse model for beta 0-thalassemia.

We have used a "plug and socket" targeting technique to generate a mouse model of beta 0-thalassemia in which both the b1 and b2 adult globin genes have been deleted. Mice homozygous for this deletion (Hbbth-3/Hbbth-3) die perinatally, similar to the most severe form of Cooley anemia in humans. Mice heterozygous for the deletion appear normal, but their hematologic indices show characteristics typical of severe thalassemia, including dramatically decreased hematocrit, hemoglobin, red blood cell counts, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, as well as dramatically increased reticulocyte counts, serum bilirubin concentrations, and red cell distribution widths. Tissue and organ damage typical of beta-thalassemia, such as bone deformities and splenic enlargement due to increased hematopoiesis, are also seen in the heterozygous animals, as is spontaneous iron overload in the spleen, liver, and kidneys. The mice homozygous for the b1 and b2 deletions should be of great value in developing therapies for the treatment of thalassemias in utero. The heterozygous animals will be useful for studying the pathophysiology of thalassemias and have the potential of generating a model of sickle cell anemia when mated with appropriate transgenic animals.

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato1

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.

Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme.

Certain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.

A 5'-upstream region of a bovine keratin 6 gene confers tissue-specific expression and hyperproliferation-related induction in transgenic mice.

Keratins, the constituents of epithelial intermediate filaments, are precisely regulated in a tissue- and development-specific manner, although little is known about the molecular mechanisms underlying this regulation. The expression pattern of keratin 6 is particularly complex, since besides being constitutively expressed in hair follicles and in suprabasal cells of a variety of internal stratified epithelia, it is induced in epidermis in both natural and artificially caused hyperproliferative situations. Therefore, the regulatory sequences controlling keratin 6 gene activity are particularly suitable for target gene expression in a tissue-specific manner. More interestingly, they can be skin-induced in transgenic animals or in gene therapy protocols, particularly those addressing epidermal hyperproliferative disorders. To delimit the regions containing these regulatory elements, different parts of the bovine keratin 6 gene linked to a beta-galactosidase reporter gene have been assayed in transgenic mice. A 9-kbp fragment from the 5' upstream region was able to provide both suprabasal tissue-specific and inducible reporter expression.

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

A 12R-lipoxygenase in human skin: Mechanistic evidence, molecular cloning, and expression

A recognized feature of psoriasis and other proliferative dermatoses is accumulation in the skin of the unusual arachidonic acid metabolite, 12R-hydroxyeicosatetraenoic acid (12R-HETE). This hydroxy fatty acid is opposite in chirality to the product of the well-known 12S-lipoxygenase and heretofore in mammals is known only as a product of cytochrome P450s. Here we provide mechanistic evidence for a lipoxygenase route to 12R-HETE in human psoriatic tissue and describe a 12R-lipoxygenase that can account for the biosynthesis. Initially we demonstrated retention of the C-12 deuterium of octadeuterated arachidonic acid in its conversion to 12R-HETE in incubations of psoriatic scales, indicating the end product is not formed by isomerization from 12S-H(P)ETE via the 12-keto derivative. Secondly, analysis of product formed from [10R-3H] and [10S-3H]-labeled arachidonic acids revealed that 12R-HETE synthesis is associated with stereospecific removal of the pro-R hydrogen from the 10-carbon of arachidonate. This result is compatible with 12R-lipoxygenase-catalyzed formation of 12R-HETE and not with a P450-catalyzed route to 12R-HETE in psoriatic scales. We cloned a lipoxygenase from human keratinocytes; the cDNA and deduced amino acid sequences share ≤50% identity to other human lipoxygenases. This enzyme, when expressed in Hela cells, oxygenates arachidonic acid to 12-HPETE, >98% 12R in configuration. The 12R-lipoxygenase cDNA is detectable by PCR in psoriatic scales and as a 2.5-kilobase mRNA by Northern analysis of keratinocytes. Identification of this enzyme extends the known distribution of R-lipoxygenases to humans and presents an additional target for potential therapeutic interventions in psoriasis.

Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta

Leptin is a 167-aa protein that is secreted from adipose tissue and is important in the regulation of energy balance. It also functions in hematopoiesis and reproduction. To assess whether leptin is involved in fetal growth and development we have examined the distribution of mRNAs encoding leptin and the leptin receptor (which has at least six splice variants) in the 14.5-day postcoitus mouse fetus and in the placenta using reverse transcription–PCR and in situ hybridization. High levels of gene expression for leptin, the leptin receptor, and the long splice variant of the leptin receptor with an intracellular signaling domain were observed in the placenta, fetal cartilage/bone, and hair follicles. Receptor expression also was detected in the lung, as well as the leptomeninges and choroid plexus of the fetal brain. Western blotting and immunocytochemistry, using specific antibodies, demonstrated the presence of leptin and leptin receptor protein in these tissues. These results suggest that leptin may play a role in the growth and development of the fetus, both through placental and fetal expression of the leptin and leptin receptor genes. In the fetus, leptin may be multifunctional and have both paracrine and endocrine effects.

Suppression of HIV replication by lymphoid tissue CD8+ cells correlates with the clinical state of HIV-infected individuals

Lymphoid tissues from asymptomatic HIV-infected individuals, as compared with symptomatic HIV-infected subjects, show limited histopathological changes and lower levels of HIV expression. In this report we correlate the control of HIV replication in lymph nodes to the non-cytolytic anti-HIV activity of lymphoid tissue CD8+ cells. Five subjects at different stages of HIV-related disease were studied and the ability of their CD8+ cells, isolated from both lymphoid tissue and peripheral blood, to inhibit HIV replication was compared. CD8+ cells from lymphoid tissue and peripheral blood of two HIV-infected long-term survivors suppressed HIV replication at a low CD8+:CD4+ cell ratio of 0.1. The CD8+ cells from the lymphoid tissue of a third asymptomatic subject suppressed HIV replication at a CD8+:CD4+ cell ratio of 0.25; the subject’s peripheral blood CD8+ cells showed this antiviral response at a lower ratio of 0.05. The lymphoid tissue CD8+ cells from two AIDS patients were not able to suppress HIV replication, and the peripheral blood CD8+ cells of only one of them suppressed HIV replication. The plasma viremia, cellular HIV load as well as the extent of pathology and virus expression in the lymphoid tissue of the two long-term survivors, were reduced compared with these parameters in the three other subjects. The data suggest that the extent of anti-HIV activity by CD8+ cells from lymphoid tissue relative to peripheral blood correlates best with the clinical state measured by lymphoid tissue pathology and HIV burden in lymphoid tissues and blood. The results add further emphasis to the importance of this cellular immune response in controlling HIV pathogenesis.

Torpor in mice is induced by both leptin-dependent and -independent mechanisms

We tested the effect of chronic leptin treatment on fasting-induced torpor in leptin-deficient A-ZIP/F-1 and ob/ob mice. A-ZIP/F-1 mice have virtually no white adipose tissue and low leptin levels, whereas ob/ob mice have an abundance of fat but no leptin. These two models allowed us to examine the roles of adipose tissue and leptin in the regulation of entry into torpor. Torpor is a short-term hibernation-like state that allows conservation of metabolic fuels. We first characterized the A-ZIP/F-1 animals, which have a 10-fold reduction in total body triglyceride stores. Upon fasting, A-ZIP/F-1 mice develop a lower metabolic rate and decreased plasma glucose, insulin, and triglyceride levels, with no increase in free fatty acids or β-hydroxybutyrate. Unlike control mice, by 24 hr of fasting, they have nearly exhausted their triglycerides and are catabolizing protein. To conserve energy supplies during fasting, A-ZIP/F-1 (but not control) mice entered deep torpor, with a minimum core body temperature of 24°C, 2°C above ambient. In ob/ob mice, fasting-induced torpor was completely reversed by leptin treatment. In contrast, neither leptin nor thyroid hormone prevented torpor in A-ZIP/F-1 mice. These data suggest that there are at least two signals for entry into torpor in mice, a low leptin level and another signal that is independent of leptin and thyroid hormone levels. Studying rodent torpor provides insight into human torpor-like states such as near drowning in cold water and induced hypothermia for surgery.

Characterization and developmental patterns of telomerase expression in plants

Telomerase activity is developmentally regulated in mammals. Here we examine telomerase activity in plants, whose development differs in fundamental ways from that of animals. Using a modified version of the telomere repeat amplification protocol (TRAP) assay, we detected an activity in extracts from carrots, cauliflower, soybean, Arabidopsis, and rice with all the characteristics expected for a telomerase synthesizing the plant telomere repeat sequence TTTAGGG. The activity was dependent on RNA and protein components, required dGTP, dATP, and dTTP, but not dCTP, and generated products with a seven nucleotide periodicity. Telomerase activity was abundant in cauliflower meristematic tissue and undifferentiated cells from Arabidopsis, soybean, and carrot suspension cultures, but was low or not detectable in a sampling of differentiated tissues from mature plants. Telomerase from cauliflower meristematic tissues exhibited relaxed DNA sequence requirements, which might reflect the capacity to form telomeres on broken chromosomes in vivo. The dramatic differences in telomerase expression and their correlation with cellular proliferation capacity mirror changes in human telomerase levels during differentiation and immortalization. Hence, telomerase activation appears to be a conserved mechanism involved in conferring long-term proliferation capacity.

Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice

To elucidate the role of neuropeptide Y (NPY)-Y1 receptor (Y1-R) in food intake, energy expenditure, and other possible functions, we have generated Y1-R-deficient mice (Y1-R−/−) by gene targeting. Contrary to our hypothesis that the lack of NPY signaling via Y1-R would result in impaired feeding and weight loss, Y1-R−/− mice showed a moderate obesity and mild hyperinsulinemia without hyperphagia. Although there was some variation between males and females, typical characteristics of Y1-R−/− mice include: greater body weight (females more than males), an increase in the weight of white adipose tissue (WAT) (approximately 4-fold in females), an elevated basal level of plasma insulin (approximately 2-fold), impaired insulin secretion in response to glucose administration, and a significant changes in mitochondrial uncoupling protein (UCP) gene expression (up-regulation of UCP1 in brown adipose tissue and down-regulation of UCP2 in WAT). These results suggest either that the Y1-R in the hypothalamus is not a key molecule in the leptin/NPY pathway, which controls feeding behavior, or that its deficiency is compensated by other receptors, such as NPY-Y5 receptor. We believe that the mild obesity found in Y1-R−/− mice (especially females) was caused by the impaired control of insulin secretion and/or low energy expenditure, including the lowered expression of UCP2 in WAT. This model will be useful for studying the mechanism of mild obesity and abnormal insulin metabolism in noninsulin-dependent diabetes mellitus.