Dietary n-3 polyunsaturated fat increases the fractional catabolic rate of medium-sized HDL particles in African green monkeys

We have previously described a novel pathway for the metabolism of HDL subfractions in which small [2 apolipoprotein (apoA-I) molecules per particle] HDL particles are converted in a unidirectional manner outside the plasma compartment to medium (3 apoA-I molecules per particle) or large (4 apoA-I molecules per particle) HDL particles, which are subsequently removed from the circulation by the liver (Colvin et al. 1999. J. Lipid Res. 40: 1782;-1792; Huggins et al. 2000. J. Lipid Res. 41: 384;-394). The purpose of the present study was to determine whether the reduction in concentration of medium HDL in African green monkeys consuming n-3 polyunsaturated versus saturated fat diets resulted from decreased in vivo production or increased catabolism. Tracer small LpA-I (HDL containing only apoA-I) were isolated, without ultracentrifugation, by gel filtration and immunoaffinity chromatography and radiolabeled. After injection, the specific activity of apoA-I in small, medium, and large HDL was determined, and the kinetic data were analyzed using our previously published multicompartmental model for HDL subfraction metabolism. We found a significant reduction of apoA-I concentration in medium HDL in the animals fed n-3 polyunsaturated fat (31.2 +/- 0.7 mg/dl) compared with animals fed saturated fat (85.4 +/- 11.9 mg/dl; P = 0.002). The production rates of apoA-I in small, medium, and large HDL were similar in both diet groups; however, there was a significant increase in the fractional catabolic rate of apoA-I in medium HDL in the animals fed n-3 polyunsaturated fat (2.188 +/- 0.501 pools/day) compared with animals fed saturated fat (0.714 +/- 0.191 pools/day; P = 0.02).We conclude that n-3 polyunsaturated fat reduces HDL cholesterol concentration by increasing the fractional catabolic rate of medium-sized HDL particles in African green monkeys.     

The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.     

Prolactin opens the sensitive period for androgen regulation of a larynx-specific myosin heavy chain gene

The larynx of Xenopus laevis is a sexually differentiated vocal organ in which male muscle is entirely fast twitch and expresses high levels of a fast twitch myosin heavy chain gene, LM. Female muscle, however, is mostly slow twitch and expresses little LM. Androgen is unable to induce expression of LM until after metamorphosis is complete. The expression of LM during metamorphic and early postmetamorphic development parallels secretion and expression of the pituitary hormone prolactin. Here, we show that exposure to prolactin is necessary to allow androgen-induced LM expression in postmetamorphic froglets. In prolactin-deprived animals, androgen-induced changes in the contractile properties of laryngeal muscle are blocked, which prevents the rapid rates of muscle contraction required for males to produce courtship songs. Thus, prolactin opens the sensitive period for androgen-induced LM expression in the larynx and controls the ability of male sex hormones to masculinize the vocal system both at the level of gene expression and vocal organ physiology.     

Regulation of monocyte survival in vitro by deposited IgG: role of macrophage colony-stimulating factor

IgG deposition at tissue sites characteristically leads to macrophage accumulation and organ injury. Although the mechanism by which deposited IgG induces tissue injury is not known, we have recently demonstrated that deposited IgG stimulates the release of IL-8 and monocyte chemoattractant protein-1 from normal human monocytes, which may drive inflammation. Since IgG also induces macrophage accumulation in these diseases, we hypothesized that deposited IgG protects monocytes from apoptosis. As an in vitro model of the effect of deposited IgG on monocyte survival, monocyte apoptosis was studied after FcgammaR cross-linking. Monocytes cultured on immobilized IgG, which induces FcgammaR cross-linking, were protected from apoptosis, whereas monocytes cultured with equivalent concentrations of F(ab')2 IgG or 50 times higher concentrations of soluble IgG, neither of which induces FcgammaR cross-linking, were not protected. Moreover, this protection was transferable, as supernatants from immobilized IgG-stimulated monocytes protected freshly isolated monocytes from apoptosis and contained functional M-CSF, a known monocyte survival factor. M-CSF mediated the monocyte survival induced by FcgammaR cross-linking, as neutralizing anti-human M-CSF Abs blocked the monocyte protection provided by either immobilized IgG or IgG-stimulated monocyte supernatants. These findings demonstrate a novel mechanism by which deposited IgG targets tissue macrophage accumulation through FcgammaR-mediated M-CSF release. This pathway may play an important role in promoting and potentiating IgG-mediated tissue injury.     

The paleoenvironment of Sivapithecus parvada

Remains of the hominoid Sivapithecus parvada and a diversity of mammalian taxa are preserved at locality Y311 (ca. 10 Ma) in the Siwalik Nagri Formation of northern Pakistan. Bovids (Bovidae, Artiodactyla) are the most abundant mammals next to tragulids (Tragulidae, Artiodactyla) at locality Y311 and provide a means for reconstructing the paleoenvironments that would have been available to Sivapithecus parvada. A functional model indicates a linkage between habitat and several femoral characters among extant bovids. Based on this model, we infer that forested habitats predominated at locality Y311 but that some less densely covered areas may also have been present. Paleoenvironments in the earlier Chinji Formation appear comparable to those at locality Y311, although the presence of a continuous canopy in the former is more certain. Thus, adaptive changes in the bovid fauna from the Chinji through the Nagri Formations appear to have preceded the shift to predominantly C4 grasslands which, based on other lines of evidence, occurred locally (and possibly globally) between 8 and 6 Ma. The paleoenvironments of locality Y311 and the Chinji Formation localities appear different from the paleoenvironment of Kenyapithecus at Fort Ternan in Kenya, where the presence of continuous canopy is unlikely. The Fort Ternan fauna is dominated by two genera of bovids. One of these is adapted to light cover while the other appears better adapted to heavy cover. Sivapithecus and Kenyapithecus lived in different ecological settings probably characterized by varying degrees of vegetative cover.     

Single-base mismatch detection based on charge transduction through DNA

High-throughput DNA sensors capable of detecting single-base mismatches are required for the routine screening of genetic mutations and disease. A new strategy for the electrochemical detection of single-base mismatches in DNA has been developed based upon charge transport through DNA films. Double-helical DNA films on gold surfaces have been prepared and used to detect DNA mismatches electrochemically. The signals obtained from redox-active intercalators bound to DNA-modified gold surfaces display a marked sensitivity to the presence of base mismatches within the immobilized duplexes. Differential mismatch detection was accomplished irrespective of DNA sequence composition and mismatch identity. Single-base changes in sequences hybridized at the electrode surface are also detected accurately. Coupling the redox reactions of intercalated species to electrocatalytic processes in solution considerably increases the sensitivity of this assay. Reporting on the electronic structure of DNA, as opposed to the hybridization energetics of single-stranded oligonucleotides, electrochemical sensors based on charge transport may offer fundamental advantages in both scope and sensitivity.     

NF-kappaB activation is required for the development of cardiac hypertrophy in vivo

In the present study, we examined whether NF-kappaB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IkappaB-alpha dominant negative mutant (IkappaB-alphaM), a superrepressor of NF-kappaB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-kappaB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-kappaB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-beta activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-kappaB activation in vivo by cardiac transfection of IkappaB-alphaM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-kappaB activity. Our results suggest that NF-kappaB activation is required for the development of cardiac hypertrophy in vivo and that NF-kappaB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.     

Interactions between glutamate transporters and metabotropic glutamate receptors at excitatory synapses in the cerebellar cortex

Five glutamate transporter genes have been identified; two of these (EAAT3 and EAAT4) are expressed in neurons and are predominantly confined to the membranes of cell bodies and dendrites. At an ultrastructural level, glutamate transporters have been shown to surround excitatory synapses in hippocampus and cerebellum [J. Neurosci. 18 (1998) 3606; J. Comp. Neurol. 418 (2000) 255]. This pattern of localization overlaps the well-described perisynaptic distribution of Group I metabotropic glutamate receptors or mGluRs [Neuron 11 (1993) 771; J. Chem. Neuroanat. 13 (1997) 77]. Both of the principal excitatory synaptic inputs to cerebellar Purkinje neurons, the parallel fiber (PF) and climbing fiber (CF) synapses, express mGluR-dependent forms of synaptic plasticity [Nat. Neurosci. 4 (2001) 467]. Prompted by the colocalization of postsynaptic glutamate transporters and mGluRs, we have examined whether glutamate uptake limits mGluR-mediated signals and mGluR-dependent forms of plasticity at PF and CF synapses in cerebellar slices. We find that, at PF and, surprisingly also at CF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release. At both PF and CF synapses, mGluR responses are strongly limited by glutamate transporters under resting conditions and are facilitated by short trains of stimuli. Nearly every Purkinje neuron expresses an mGluR-mediated synaptic current upon inhibition of glutamate transport. Global applications of glutamate achieved by photolysis of chemically caged glutamate yield similar results and argue that the colocalized transporters can effectively limit glutamate access to the mGluRs even in the face of such a large amount of transmitter. We hypothesize that neuronal glutamate transporters and Group I mGluRs located in the perisynaptic space interact to sense and then regulate the amount of glutamate escaping excitatory synapses. This hypothesis is currently being tested using electrophysiological methods and the introduction of optically tagged glutamate transporter proteins. In the brain, synaptic signals are terminated mainly by neurotransmitter transporters. Families of genes encoding transporters for the major neurotransmitters (dopamine, GABA, glutamate, glycine, norepinephrine and 5-HT) have been identified. Although transporters serve as targets for important classes of therapeutic drugs (e.g. selective serotonin reuptake inhibitors) and drugs of abuse (amphetamine, cocaine), little is known about how they operate at a molecular level or contribute to synaptic transmission.