A fish oil diet inhibits amyloid P component (AP) acute phase responses in arthritis susceptible mice

Amyloid P component (AP) bears close homology with C-reactive protein and behaves as an acute phase reactant in the plasma of mice but not in man. Our aim was to determine whether AP is influenced by diet, gender, and arthritis severity in a murine model of arthritis. B10.RIII mice were segregated according to gender and diet at 8 wk of age: the source of fat was either corn oil, fish oil, or beef tallow (5% by weight). Four weeks later, each mouse was immunized with 100 micrograms fetal bovine type II collagen, and the incidence and severity of arthritis was noted at weekly intervals. AP was measured by competitive ELISA in plasma taken 5 wk and 15 wk after immunization. AP levels were less in fish oil fed males and females. Under all conditions tested AP levels of females were greater than in males. There was a negative correlation between AP levels and the severity of arthritis. We conclude from these data that although AP levels cannot be used as indices of arthritis severity, there are significant dietary and gender effects on AP concentrations as long as 15 wk after immunization with type II collagen.     

Divergent responses of Atlantic cod to ocean acidification and food limitation

In order to understand the effect of global change on marine fishes, it is imperative to quantify the effects on fundamental parameters such as survival and growth. Larval survival and recruitment of the Atlantic cod (Gadus morhua) were found to be heavily impaired by end‐of‐century levels of ocean acidification. Here, we analysed larval growth among 35–36 days old surviving larvae, along with organ development and ossification of the skeleton. We combined CO₂ treatments (ambient: 503 µatm, elevated: 1,179 µatm) with food availability in order to evaluate the effect of energy limitation in addition to the ocean acidification stressor. As expected, larval size (as a proxy for growth) and skeletogenesis were positively affected by high food availability. We found significant interactions between acidification and food availability. Larvae fed ad libitum showed little difference in growth and skeletogenesis due to the CO₂ treatment. Larvae under energy limitation were significantly larger and had further developed skeletal structures in the elevated CO₂ treatment compared to the ambient CO₂ treatment. However, the elevated CO₂ group revealed impairments in critically important organs, such as the liver, and had comparatively smaller functional gills indicating a mismatch between size and function. It is therefore likely that individual larvae that had survived acidification treatments will suffer from impairments later during ontogeny. Our study highlights important allocation trade‐off between growth and organ development, which is critically important to interpret acidification effects on early life stages of fish.     

African Y chromosome and mtDNA divergence provides insight into the history of click languages

BACKGROUND: About 30 languages of southern Africa, spoken by Khwe and San, are characterized by a repertoire of click consonants and phonetic accompaniments. The Jumid R:'hoansi (!Kung) San carry multiple deeply coalescing gene lineages. The deep genetic diversity of the San parallels the diversity among the languages they speak. Intriguingly, the language of the Hadzabe of eastern Africa, although not closely related to any other language, shares click consonants and accompaniments with languages of Khwe and San. RESULTS: We present original Y chromosome and mtDNA variation of Hadzabe and other ethnic groups of Tanzania and Y chromosome variation of San and peoples of the central African forests: Biaka, Mbuti, and Lisongo. In the context of comparable published data for other African populations, analyses of each of these independently inherited DNA segments indicate that click-speaking Hadzabe and Jumid R:'hoansi are separated by genetic distance as great or greater than that between any other pair of African populations. Phylogenetic tree topology indicates a basal separation of the ancient ancestors of these click-speaking peoples. That genetic divergence does not appear to be the result of recent gene flow from neighboring groups. CONCLUSIONS: The deep genetic divergence among click-speaking peoples of Africa and mounting linguistic evidence suggest that click consonants date to early in the history of modern humans. At least two explanations remain viable. Clicks may have persisted for tens of thousands of years, independently in multiple populations, as a neutral trait. Alternatively, clicks may have been retained, because they confer an advantage during hunting in certain environments.     

Acyl-CoA: glycine N-acyltransferase: in vitro studies on the glycine conjugation of straight- and branched-chained acyl-CoA esters in human liver

Apparent kinetic constants (Km and Vmax values) were determined for human liver acyl-CoA: glycine acyltransferase (glycine-N-acylase) towards isobutyryl-CoA, 2-methyl butyryl-CoA, isovaleryl-CoA, butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, and decanoyl-CoA. These acyl-CoA esters were selected because of their relevance to the human diseases with cellular accumulation of these esters, i.e., especially to metabolic defects in the acyl-CoA dehydrogenation steps of the branched-chain amino acids, lysine, 5-hydroxy lysine, tryptophan, and fatty acid oxidation pathways. With the acyl-CoA ester as the fixed substrate, the Km value for glycine ranged from 0.5 to 2.9 mole/liter, and with glycine as fixed substrate, the Km values for the acyl-CoA esters varied from 0.3 to 5.6 mmole/liter. It is concluded that the substrate concentration is decisive for the glycine conjugate formation and that the occurrence in urine of acylglycines reflects an intramitochondrial accumulation of the corresponding acyl-CoA ester.     

Targeted inactivation of Galpha(i) does not alter cardiac function or beta-adrenergic sensitivity

Inhibitory Galpha(i) protein increases in the myocardium during hypertrophy and has been associated with beta-adrenergic receptor (beta-AR) desensitization, contractile dysfunction, and progression of cardiac disease. The role of Galpha(i) proteins in mediating basal cardiac function and beta-AR response in nonpathological myocardium, however, is uncertain. Transgenic mice with targeted inactivation of Galpha(i2) or Galpha(i3) were examined for in vivo cardiac function with the use of conscious echocardiography and for ex vivo cardiac response to inotropic stimulation with the use of Langendorff blood-perfused isolated hearts and adult ventricular cardiomyocytes. Echocardiography revealed that percent fractional shortening and heart rate were similar among wild-type, Galpha(i2)-null, and Galpha(i3)-null mice. Comparable baseline diastolic and contractile performance was also observed in isolated hearts and isolated ventricular myocytes from wild-type mice and mice lacking Galpha(i) proteins. Isoproterenol infusion enhanced diastolic and contractile performance to a similar degree in wild-type, Galpha(i2)-null, and Galpha(i3)-null mice. These data demonstrate no observable role for inhibitory G proteins in mediating basal cardiac function or sensitivity to beta-AR stimulation in nonpathological myocardium.     

Structure determination of T cell protein-tyrosine phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130-132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme.     

Activation of Rap1B by G(i) family members in platelets

It has become increasingly appreciated that receptors coupled to G(alpha)(i) family members can stimulate platelet aggregation, but the mechanism for this has remained unclear. One possible mediator is the small GTPase, Rap1, which has been shown to contribute to integrin activation in several cell lines and to be activated by a calcium-dependent mechanism in platelets. Here, we demonstrate that Rap1 is also activated by G(alpha)(i) family members in platelets. First, we show that platelets from mice lacking the G(alpha)(i) family member G(alpha)(z) (which couples to the alpha(2A) adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking G(alpha)(i2), which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack G(alpha)(q) show no decrease in the ability to activate Rap1 in response to epinephrine but show a partial reduction in ADP-stimulated Rap1 activation. This result, combined with studies of human platelets treated with ADP receptor-selective inhibitors, indicates that ADP-stimulated Rap1 activation in human platelets is dependent on both the G(alpha)(i)-coupled P2Y12 receptor and the G(alpha)(q)-coupled P2Y1 receptor. G(alpha)(i)-dependent activation of Rap1 in platelets does not appear to be mediated by enhanced intracellular calcium release because no increase in intracellular calcium concentration was detected in response to epinephrine and because the calcium response to ADP was not diminished in platelets from the G(alpha)(i2)-/- mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the G(beta)(gamma)-activated form of his enzyme (PI3Kgamma), we show that G(i)-mediated Rap1 activation is PI3K-dependent. In summary, activation of Rap1 can be stimulated by G(alpha)(i)- and PI3K-dependent mechanisms in platelets and by G(q)- and Ca(2+)-dependent mechanisms, both of which may play a role in promoting platelet activation.     

Mouse hepatocyte synthesis and induction of the acute phase reactant: serum amyloid P-component

A methodology for obtaining reproducible in vitro induction of the synthesis of the acute phase reactant serum amyloid P-component (SAP) by purified mouse hepatocytes was established. Optimal hepatocyte culture conditions for the induction and synthesis of SAP required certain hormones, a substratum for cell attachment, and activated macrophages. Leibowitz L15 medium had to be supplemented with dexamethasone, indomethacin, insulin, glucose, and fetal bovine serum. Purified mouse IL 1 could substitute for activated macrophages in the induction of SAP. Hepatocytes were allowed to adhere to a collagen matrix to enhance both cell viability and SAP synthesis induced by IL 1. Elicited macrophages cultured with hepatocytes were capable of augmenting SAP synthesis in the presence of IL 1.     

Thyroid hormone-dependent gene expression as a biomarker of short-term 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) exposure in European common frog (Rana temporaria) tadpoles

The effects on thyroid hormone-dependent gene biomarker responses of the persistent organochlorine pesticide metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) were investigated after exposure of 4-week-old European common frog (Rana temporaria) (stage 36) tadpoles to two (0.001 and 0.01 ppm) DDE concentrations. Total body weight, total length, and tail length and width increased after 3-day exposure to DDE. Expression patterns of genes encoding for growth hormone, thyroid-stimulating hormone (TSHβ) and thyroid hormone receptor (TRα and TRβ) isoforms were evaluated in the head, body and tail regions using a validated real-time polymerase chain reaction (PCR) method. The mRNA expression of growth hormone in the body, and TSHβ in the head showed significant DDE concentration-dependent decreases. While DDE caused variable effects on TRα mRNA steady-state, the expression of TRβ was significantly decreased in the tail by DDE in a concentration-specific manner. The effect of DDE exposure on TRβ mRNA expression showed a negative correlation with tail length and width during the exposure period. The unique pattern of a DDE-induced decrease of tail TRβ expression probably reflects the significant role of this thyroid hormone receptor isoform in tail re-absorption and overall metamorphosis in anuran species. Therefore, the present study shows that the evaluation of thyroid hormone-dependent genes may represent quantitative biomarkers of acute exposure to organochlorine pesticides in anuran species during critical developmental periods such as metamorphosis. Given the widespread environmental levels of DDT and its metabolites, these pollutants will remain a subject of concern and their effects on anuran species should be studied in more detail.