Function of the human immunodeficiency virus types 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA.

The interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region in env mRNA (the Rev-responsive element [RRE]) mediates the export of structural mRNAs from the nucleus to the cytoplasm. We demonstrated that unlike HIV-1 Rev, which functions with both the HIV-1 and HIV-2 RREs, HIV-2 Rev functions only with the HIV-2 RRE. Rev-RRE binding studies suggested that the lack of nonreciprocal complementation stems from the inability of HIV-2 Rev to interact with HIV-1 RRE RNA. Maintenance of RNA secondary structure, rather than the primary nucleotide sequence, appeared to be the major determinant for interaction of both HIV-1 and HIV-2 Rev with the HIV-2 RRE. Moreover, the binding domain of the HIV-2 RRE recognized by HIV-1 Rev was dissimilar to the binding domain of the HIV-1 RRE, in terms of both secondary structure and primary nucleotide sequence. Our results support the hypothesis that function of HIV Rev proteins and possibly the functionally similar Rex proteins encoded by the human T-cell leukemia viruses (HTLVs) HTLV-I and HTLV-II is controlled by the presence of RNA secondary structure generated within the RRE RNA.     

Sphingosine Kinase 1 Is Regulated by Peroxisome Proliferator-activated Receptor α in Response to Free Fatty Acids and Is Essential for Skeletal Muscle Interleukin-6 Production and Signaling in Diet-induced Obesity

We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3)_. Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1^−/− mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1^−/− mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.     

Environment and feeding change the ability of heart rate to predict metabolism in resting Steller sea lions (<Emphasis Type="Italic">Eumetopias jubatus</Emphasis>)

The ability to use heart rate (fh) to predict oxygen consumption rates ( [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} ) in Steller sea lions and other pinnipeds has been investigated in fasting animals. However, it is unknown whether established fh: [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} relationships hold under more complex physiological situations, such as when animals are feeding or digesting. We assessed whether fh could accurately predict [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} in trained Steller sea lions while fasting and after being fed. Using linear mixed-effects models, we derived unique equations to describe the fh: [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} relationship for fasted sea lions resting on land and in water. Feeding did not significantly change the fh: [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} relationship on land. However, Steller sea lions in water displayed a different fh: [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} relationship after consuming a 4-kg meal compared with the fasting condition. Incorporating comparable published fh: [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} data from Steller sea lions showed a distinct effect of feeding after a 6-kg meal. Ultimately, our study illustrated that both feeding and physical environment are statistically relevant when deriving [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} from telemetered fh, but that only environment affects the practical ability to predict metabolism from fh. Updating current bioenergetic models with data gathered using these predictive fh: [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} equations will yield more accurate estimates of metabolic rates of free-ranging Steller sea lions under a variety of physiological, behavioral, and environmental states.     

Synchronization of Isolated Downstates (K-Complexes) May Be Caused by Cortically-Induced Disruption of Thalamic Spindling

Sleep spindles and K-complexes (KCs) define stage 2 NREM sleep (N2) in humans. We recently showed that KCs are isolated downstates characterized by widespread cortical silence. We demonstrate here that KCs can be quasi-synchronous across scalp EEG and across much of the cortex using electrocorticography (ECOG) and localized transcortical recordings (bipolar SEEG). We examine the mechanism of synchronous KC production by creating the first conductance based thalamocortical network model of N2 sleep to generate both spontaneous spindles and KCs. Spontaneous KCs are only observed when the model includes diffuse projections from restricted prefrontal areas to the thalamic reticular nucleus (RE), consistent with recent anatomical findings in rhesus monkeys. Modeled KCs begin with a spontaneous focal depolarization of the prefrontal neurons, followed by depolarization of the RE. Surprisingly, the RE depolarization leads to decreased firing due to disrupted spindling, which in turn is due to depolarization-induced inactivation of the low-threshold Ca²⁺ current (I_T). Further, although the RE inhibits thalamocortical (TC) neurons, decreased RE firing causes decreased TC cell firing, again because of disrupted spindling. The resulting abrupt removal of excitatory input to cortical pyramidal neurons then leads to the downstate. Empirically, KCs may also be evoked by sensory stimuli while maintaining sleep. We reproduce this phenomenon in the model by depolarization of either the RE or the widely-projecting prefrontal neurons. Again, disruption of thalamic spindling plays a key role. Higher levels of RE stimulation also cause downstates, but by directly inhibiting the TC neurons. SEEG recordings from the thalamus and cortex in a single patient demonstrated the model prediction that thalamic spindling significantly decreases before KC onset. In conclusion, we show empirically that KCs can be widespread quasi-synchronous cortical downstates, and demonstrate with the first model of stage 2 NREM sleep a possible mechanism whereby this widespread synchrony may arise.     

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.     

Persistent but reversible coronary microvascular dysfunction after bypass grafting

The effect of coronary artery bypass grafting (CABG) on absolute myocardial blood flow (MBF) has not been investigated previously. MBF (ml. min(-1). g(-1)) was measured at rest and during hyperemia (0.56 mg/kg iv dipyridamole) using H(2)(15)O and positron emission tomography in eight patients with three-vessel disease before surgery and 1 and 6 mo after full revascularization. Baseline MBF was 0.87 +/- 0.12 preoperatively and 1.04 +/- 0.14 and 0.95 +/- 0.13 at 1 and 6 mo after CABG, respectively (P < 0.05, 6 mo vs. preoperatively). Hyperemic MBF was 1.36 +/- 0.28 preoperatively and increased to 1.98 +/- 0.50 and 2.45 +/- 0.64 at 1 and 6 mo after CABG, respectively (P < 0.01, 6 mo vs. preoperatively). Coronary vasodilator reserve (hyperemic/baseline MBF) increased from 1.59 +/- 0.40 preoperatively to 1.93 +/- 0.13 and 2.57 +/- 0.49 at 1 and 6 mo, respectively (P < 0.05, 6 mo vs. preoperatively). Minimal (dipyridamole) coronary resistance (mmHg. min. g(-1). ml(-1)) fell progressively from 59.37 +/- 14.56 before surgery to a nadir of 35. 76 +/- 10.12 at 6 mo after CABG (P < 0.01 vs. preoperatively). The results of the present study confirm that CABG improves coronary vasodilator reserve progressively as a result of reduction in minimal coronary resistance. These data suggest persistent microvascular dysfunction that recovers slowly after surgery.