Stimulation of DNA synthesis in human fibroblasts by a human nonsuppressible insulin-like protein (NSILP).

When nonsuppressible insulin-like protein (NSILP) isolated and purified from human serum was added at concentrations of 5 and 50 ug/ml to cultures of human dermal fibroblasts, both cell proliferation and DNA synthesis were enhanced. However, NSILP, 50 ug/ml, had no effect on glucose uptake. In contrast, insulin, 40 ng/ml (1.0 mU/ml), had no effect on cell proliferation or DNA synthesis, but stimulated glucose uptake. These observations suggest that human NSILP may play an important role in tissue repair or growth by enhancing fibroblast proliferation, but not a significant glucoregulatory role.     

Disruption of the TGF-beta pathway and modeling human cancer in mice

There is considerable complexity underlying the mechanisms through which the TGF-beta signaling pathway regulates the initiation and progression of cancer. Analysis of this pathway and the role that it plays in human malignancy continues to elucidate novel mechanisms through which various genetic and epigenetic events subvert the controls that TGF-beta exerts over cell growth, differentiation, and malignant transformation. Modeling these events in the mouse represents an important goal, as the relevant preclinical models are essential not only for improving our understanding of the role of the TGF-beta pathway in the molecular pathogenesis of cancer, but also as tools for evaluating the impact of novel therapeutics on TGF-beta signaling and the role they may play in the prevention and treatment of malignancies. Here, we consider highlights from a number of in vivo murine model systems and relate a few of the significant observations to what we know about TGF-beta signaling in human cancer.     

Mitochondrial metabolism of hydroxocobalamin: synthesis of adenosylcobalamin by intact rat liver mitochondria.

When free hydroxocobalamin (vitamin B₁₂) is added in vitro to a suspension of intact rat liver mitochondria in the presence of a source of both reducing equivalents and ATP, adenosylcobalamin synthesis is observed. This synthetic process is not dependent on electron transport or oxidative phosphorylation and is not detected when cyanocobalamin is substituted for hydroxocobalamin. Adenosylcobalamin synthesis is linear with time for at least 10 min and with hydroxocobalamin concentration up to 37 nm. At the latter concentration of hydroxocobalamin, the rate of synthesis at 37 °C is 0.26 pmol/min/mg of protein. Only part (<30%) of the newly synthesized adenosylcobalamin is bound to the mitochondrial cobalamin binding protein, whereas most (90%) of the concurrently accumulated hydroxocobalamin is bound. On the other hand, when adenosylcobalamin is added to a suspension of intact mitochondria, it is accumulated at a rate similar to that for hydroxocobalamin, and is bound to the mitochondrial binding protein to a similar extent. These findings indicate that rat liver mitochondria contain all of the enzymatic components necessary to convert hydroxocobalamin to adenosylcobalamin, the coenzyme for the mitochondrial enzyme methylmalonyl CoA mutase.     

Redox modulation of reductase and phosphatase activities in human erythrocytes

Summary We have previously reported that ferricyanide reductase activity in human erythrocytes depended on glycolysis and could be modulated by several compounds including oxidants and hormones like insulin. Insulin could activate glycolysis, probably as a consequence of tyrosine phosphorylation of protein band 3, implicating phosphorylation reactions as an important signal for activation of the reductase by insulin. Reversible phosphorylation of cellular proteins is also believed to play a key role in the action of insulin. Cytosolic acid phosphatase activity has been found in human erythrocytes. To further extend initial reports, we studied the effect of modulators on the cytosolic erythrocyte acid phosphatase. Mild oxidants like ferricyanide (1 mM), vanadate (1 mM), Mn²⁺ (0.5 and 1 mM), and phenylarsine oxide (10 and 100 M) inhibited the phosphatase activity. Similarly, insulin at concentrations that stimulate ferricyanide reduction (500, 1000 IU/ml) inhibited the activity of the phosphatase enzyme. The overall results indicated that oxidants are able to inhibit the acid phosphatase and stimulate the redox enzyme. In addition, a significant negative correlation (r = –0.400; P = 0.006) was observed between phosphatase and reductase activities. The observations discussed here, together with previous ones, emphasize that a close association between reductase and phosphatase enzymes may exist and also suggest a role for redox reactions in tyrosine phosphorylation/dephosphorylation-mediated signal transduction pathways.     

Interference by methylcobalamin analogues with synthesis of cobalamin coenzymes in human lymphocytes in vitro.

1. 72 h uptake of cyano[57Co]cobalamin and formation of 57Co-labelled methylcobalamin, adenosylcobalamin and hydroxocobalamin has been estimated with and without the addition of methylcobalamin analogues in phytohaemagglutinin-stimulated lymphocytes from healthy human subjects. 2. Difluorochloromethylcobalamin reduced cell uptake of cyanocobalamin and caused a disproportionate reduction in synthesis of adenosylcobalamin. 3. Methylcobalamin-palladium trichloride reduced cell uptake of cyanobalamin more effectively than did difluorochloromethylcobalamin and reduced the formation of methylcobalamin, adenosylcobalamin and hydroxocobalamin in proportion. 4. The results suggest that in addition to inhibiting uptake of cyanocobalamin, one or both compounds may have interfered directly with the mechanism of synthesis of the cobalamin coenzymes.     

Nuclear beta-catenin localization is not sufficient for canonical Wnt signaling activation in human meianoma cell lines

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called beta-catenin. Mutations promoting beta-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers, but rarely observed in melanomas. Nevertheless, beta-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why, the aim of the investigation was to elucidate the relation between beta-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular beta-catenin localization, and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of beta-catenin does not always correspond to active status canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear beta-catenin canonical Wnt signaling can't be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, the pathway-targeted potential antineoplastic therapy requires the formation of a "molecular pattern of cancer" for localization of the defect in Wnt signaling cascade in the each case.     

In vivo suppression of the primary immune response by a species of low density serum lipoprotein.

An apparent subspecies of normal human serum low density lipoprotein (LDL-In) has been identified with suppressive activity for early or facilitating events of human lymphocyte mitogen and allogenic cells stimulation in vitro. This report describes the effects of in vivo administration of LDL-In on the mouse anti-SRBC immune response. Human LDL-In is not species specific and was capable of suppressing the in vivo mouse anti-sheep erythrocyte (SRBC) hemagglutination response by 88% after the administration of 500 to 600 mug LDL-In IV, whereas human serum high density lipoproteins and fibrinogen had no effect. Maximal suppression occurred only when LDL-In was injected 24 to 48 hr before antigen administration. Simultaneous or subsequent injection of LDL-In had no effect. The activity of LDL-In was influenced by antigen dose and maximal at low antigen doses. The number of splenic plaque-forming cells was also reduced indicating a suppression of the clonal expansion of primary B cells to antibody-secreting cells rather than only suppression of antibody synthesis by differentiated B cells and their progeny. These observations suggest the hypothesis that endogenous LDL-In could play an important immunoregulatory role in the maintenance of immune homeostasis and the "natural" suppression of non-productive lymphocyte proliferation.     

Influence of Fruit Availability on Fruit Consumption in a Generalist Primate,the Rhesus Macaque <Emphasis Type="Italic">Macaca mulatta</Emphasis>

Fluctuations in resource availability occur in all ecosystems. To survive, species must alter their foraging strategies according to the quantity, quality, and distribution of available food. The rhesus macaque (Macaca mulatta), a commensal primate, is considered a generalist omnivore and very few studies have addressed how its feeding strategies change with respect to resource availability. We examined dietary diversity and frugivory levels in a group of rhesus macaques at the Buxa Tiger Reserve in northern India across one year. Using behavioural observations of diet and phenological monitoring, we found that although rhesus macaques fed on 107 food items including leaves, flowers, fruits, seeds, and insects, fruits made up ca. 74% of their diet. Fruit consumption correlated positively with fruit availability, but fruit preference appeared to play an important role; 16% of all the fruit species they fed on accounted for >50% of all fruit feeding observations. We suggest that afforestation programs involving preferred fruit species at the agricultural land–forest interface would prevent forest groups of rhesus macaques from gravitating toward human habitations and reduce conflict over anthropogenic resources. We further propose that the movement of certain primates in the direction of human habitations may be contingent on resource availability and food preference rather than an inherent propensity to gravitate to anthropogenic areas.     

Ultracytochemical localizations of adenylate cyclase,guanylate cyclase and cyclic 3′,5′-nucleotide phosphodiesterase activity on the trophoblast in the human placenta

Summary Ultracytochemical localizations of cyclic nucleotide-metabolizing enzymes, namely adenylate cyclase (AC), guanylate cyclase (GC) and cyclic 3,5-nucleotide phosphodiesterase (PDE), have been demonstrated in the human term placenta. AC activity was found positive on the basal plasma membrane of the syncytiotrophoblast and on the pinocytotic vesicle of the fetal capillary endothelial cell. GC activity was observed to be strong on the plasma membrane of the microvilli of the syncytiotrophoblast. The cAMP PDE activity was shown positive both on the basal plasma membrane and on the microvillous membrane, while cGMP PDE activity was exclusively confined to the microvilli of the syncytiotrophoblast. These observations suggest that the syncytiotrophoblast plays an important role in the cyclic nucleotide metabolism in the human term placenta and that there might be significant functional differences between its basal plasma membrane and its microvillous membrane.     

Endogenous zinc concentrations in cysteamine-induced duodenal ulcers in the rat

Exogenously administered zinc compounds have been shown to possess anti-ulcer activity against a wide variety of ulcerogenic agents, both in laboratory animal models and in human peptic ulcer disease. However, a strong possibility exists that endogenous zinc may also play an important role during noxious events by various mechanisms. Therefore, the aim of this study was to focus on the changes of endogenous zinc serum and tissue concentrations in cysteamine-induced duodenal lesions. We used atomic absorption spectropho-tometry to determine the tissue and serum concentrations of zinc in normal (control) rats and those with cysteamine-induced duodenal ulcers. The results obtained in this study indicated that the onset, development and spontaneous healing of ulcer lesions were associated with certain shifts in zinc serum and tissue concentrations. Prior to ulcer formation, a significant increase was noted in serum zinc values. With the onset of duodenal lesions, zinc serum concentrations significantly decreased, while there was a significant increase in duodenal tissue concentrations when compared to healthy control animals. Zinc tissue concentrations decreased and returned to starting values by the end of the first week of spontaneous healing. This decrease in zinc tissue concentration corresponded to the healing rate of the duodenal ulcers. Serum zinc concentrations also returned to starting values within the first week period. These observations indicate and confirm that zinc could play an important role in duodenal ulcer disease and represent a natural defense system in the body.     

Elevation of histidinoalanine in calcified human aortas

The cross-links histidinoalanine (HA); pyridinoline (Pyr); desmosine (Des); and isodesmosine (Ides) in human atherosclerotic aortas were studied. Only HA showed a significant increase in calcified aortas, with a high concentration in the insoluble "mineralized" fraction, which was separated out after treatment of tissues with pronase E. The cross-links composition was similar among "mineralized" fractions prepared from tissues of varying degrees of calcification: values were 2.40; 0.10; 0.17; and 0.16 moles per 1000 moles of amino acid residues for HA; Pyr; Des; and Ides, respectively. The findings suggest that the HA-containing peptide may play an important role in the calcification process of aortic tissues.     

Modulation of the PC12 cell response to nerve growth factor by antisense oligonucleotide to amyloid precursor protein

Summary 1. The amyloid precursor protein (APP) is widely distributed among eukaryotic cells, however, its precise role in cellular functioning is not fully clarified. APP is glycoprotein membrane constituent and it may facilitate membrane associated functions.2. The aim of the present study was to examine the possibility that APP may play a role in mediating cellular trophic responses. The methods made use of an antisense oligonucleotide that was prepared to the 5 terminus of APP and shown specifically to reduce the level of APP isoforms.3. In sequential mixing experiments it was observed that the APP antisense oligonucleotide did not significantly modify the trophic response of PC12 cells pretreated with nerve growth factor (NGF). However, pretreatment of cells with the antisense oligonucleotide diminished NGF-induced increases in cellular size and neurite length.4. These observations suggest that APP may play a role in modulating the trophic response. The combined use of APP antisense oligonucleotides and neurotrophic agents may find clinical utility in the treatment of Alzheimertype dementia since it is known that NGF normally causes increases in APP levels.     

Inhibition of HERG potassium channels by celecoxib and its mechanism

Background

Celecoxib (Celebrex), a widely prescribed selective inhibitor of cyclooxygenase-2, can modulate ion channels independently of cyclooxygenase inhibition. Clinically relevant concentrations of celecoxib can affect ionic currents and alter functioning of neurons and myocytes. In particular, inhibition of Kv2.1 channels by celecoxib leads to arrhythmic beating of Drosophila heart and of rat heart cells in culture. However, the spectrum of ion channels involved in human cardiac excitability differs from that in animal models, including mammalian models, making it difficult to evaluate the relevance of these observations to humans. Our aim was to examine the effects of celecoxib on hERG and other human channels critically involved in regulating human cardiac rhythm, and to explore the mechanisms of any observed effect on the hERG channels.

Methods and Results

Celecoxib inhibited the hERG, SCN5A, KCNQ1 and KCNQ1/MinK channels expressed in HEK-293 cells with IC₅₀s of 6.0 µM, 7.5 µM, 3.5 µM and 3.7 µM respectively, and the KCND3/KChiP2 channels expressed in CHO cells with an IC₅₀ of 10.6 µM. Analysis of celecoxib''s effects on hERG channels suggested gating modification as the mechanism of drug action.

Conclusions

The above channels play a significant role in drug-induced long QT syndrome (LQTS) and short QT syndrome (SQTS). Regulatory guidelines require that all new drugs under development be tested for effects on the hERG channel prior to first administration in humans. Our observations raise the question of celecoxib''s potential to induce cardiac arrhythmias or other channel related adverse effects, and make a case for examining such possibilities.     

Cell-by-cell scanning of whole mitochondrial genomes in aged human heart reveals a significant fraction of myocytes with clonally expanded deletions

Quantitative information on the cell-to-cell distribution of all possible mitochondrial DNA (mtDNA) mutations in young and aged tissues is needed to assess the relevance of these mutations to the aging process. In the present study, we used PCR amplification of full-length mitochondrial genomes from single cells to scan human cardiomyocytes for all possible large deletions in mtDNA. Analysis of more than 350 individual cells that were derived from three middle-aged and four centenarian donors demonstrates that while most of the cells contain no deletions, in certain cardiomyocytes a significant portion of the mtDNA molecules carried one particular deletion. Different affected cells contained different deletions. Although similar numbers of cells were screened for each donor, these deletion-rich cells were found only in the hearts of old donors, where they occurred at a frequency of up to one in seven cells. These initial observations demonstrate the efficiency of the method and indicate that mitochondrial mutations have the potential to play an important role in human myocardial aging.     

Activation of the human complement system by cholesterol-rich and PEGylated liposomes-modulation of cholesterol-rich liposome-mediated complement activation by elevated serum LDL and HDL levels

Intravenously infused liposomes may induce cardiopulmonary distress in some human subjects, which is a manifestation of "complement activation-related pseudoallergy." We have now examined liposome-mediated complement activation in human sera with elevated lipoprotein (LDL and HDL) levels, since abnormal or racial differences in serum lipid profiles seem to modulate the extent of complement activation and associated adverse responses. In accordance with our earlier observations, cholesterol-rich (45 mol% cholesterol) liposomes activated human complement, as reflected by a significant rise in serum level of S-protein-bound form of the terminal complex (SC5b-9). However, liposome-induced rise of SC5b-9 was significantly suppressed when serum HDL cholesterol levels increased by 30%. Increase of serum LDL to levels similar to that observed in heterozygous familial hypercholesterolemia also suppressed liposome-mediated SC5b-9 generation considerably. While intravenous injection of cholesterol-rich liposomes into pigs was associated with an immediate circulatory collapse, the drop in systemic arterial pressure following injection of liposomes preincubated with human lipoproteins was slow and extended. Therefore, surface-associated lipoprotein particles (or apolipoproteins) seem to lessen liposome-induced adverse haemodynamic changes, possibly as a consequence of suppressed complement activation in vivo. PEGylated liposomes were also capable of activating the human complement system, and the presence of surface projected methoxypoly(ethylene glycol) chains did not interfere with generation of C3 opsonic fragments. We also show that poly(ethylene glycol) is not responsible for PEGylated liposome-mediated complement activation. The net anionic charge on the phosphate moiety of the phospholipid-mPEG conjugate seemed to play a critical role in activation of both the classical and alternative pathways of the complement system.     

Molecular characterization of the T cell repertoire using immunoscope analysis and its possible implementation in clinical practice

T lymphocytes play a central role in the pathogenesis of a large number of human conditions including autoimmunity and graft rejection. Although T cells are key players in mounting immune responses, the assessment of T cell repertoires has yet to find an important role in clinical decision making. In this review, we discuss the "immunoscope" technique and its potential diagnostic role in a variety of clinical scenarios. This is an RT-PCR based approach that subdivides a bulk T cell population (i. e. from blood, lymph, spleen, or tissue) into approximately 2800 groups based upon rearranged variable beta (Vbeta)/joining beta (Jbeta) gene segments and the resulting length of the T cell receptor's (TCR's) third complementarity determining region (CDR-3). This extensive subdivision, or focusing, allows clonal expansions to be directly observed. Such a fine-tuned analysis has revealed previously unappreciated aspects of the T cell repertoire. For instance, an antigen-specific immune response can be divided into both public and non-public components. The non-public repertoire contains the majority of the expanding T cells which are unique to the individual (private), or shared by only some (semi-private), while "public" T cells can be found responding to the antigenic determinant in every individual. Although they are often a minority of the response, the public T cell repertoire seems to play a more important role in defining, as well as driving, the overall immune phenotype in the animal. Immunoscope analysis has identified public and non-public responses in human pathologies, such as multiple sclerosis. The ability to characterize the driver T cells dictating the state of immunity/autoimmunity in individual patients will be an important step towards understanding autoimmunity and designing effective treatment for a variety of conditions including rheumatoid arthritis and multiple sclerosis. We review the current literature involving public and non-public repertoires and discuss the prospect that immunoscope analysis may play a central role in the study and perhaps the management of human autoimmune diseases, and cancer.     

Stimulation of collagen synthesis in fibroblast cultures by superoxide.

Exposure of diploid fetal human fibroblasts (IMR-90) to superoxide generated by dihydroxyfumarate resulted in increased collagen synthesis. The synthesis of type III collagen was stimulated to a greater extent than the synthesis of type I collagen. The stimulation of collagen synthesis was abolished by superoxide dismutase. Our observations suggest that superoxide may play a role in the regulation of collagen synthesis and may modulate differential collagen gene expression. These observations may explain the increased synthesis of collagen in tissues following inflammation or exposure to oxidant conditions.     

Clouded leopard (<Emphasis Type="Italic">Neofelis diardi</Emphasis>) predation on proboscis monkeys (<Emphasis Type="Italic">Nasalis larvatus</Emphasis>) in Sabah,Malaysia

In this study, we have reported two direct observations of individuals from a one-male group of proboscis monkeys (Nasalis larvatus) being killed by clouded leopards (Neofelis diardi) in the riverine forest along the Menanggul river, a tributary of the Kinabatangan river in Sabah, Malaysia. One of the two individuals was an infant female and the other was a juvenile female. Based on literature reviews and the observations reported here, we suggest that clouded leopard and crocodile might be significant potential predators of proboscis monkeys of any age or sex and that predation threats elicit the monkeys' anti-predator strategies. Moreover, the observations of the monkeys' behaviour when the group is attacked by a predator suggest that the adult males in one-male groups play an important role as protectors.