Proteomic analysis of plasma membrane lipid rafts of HL-60 cells

Neutrophils acquire phagocytic activity as they differentiate. Recently, plasma membrane lipid rafts have been shown to play important roles in the process of phagocytosis in neutrophils. To characterize the proteins involved in phagocytosis and to elucidate the process by which they acquire phagocytic activity, we investigated by nano-LC-MS/MS analysis the changes in protein composition of plasma membrane lipid rafts during DMSO-induced differentiation of the human leukemia cell line HL-60 cells into neutrophilic lineage. Based on the spectrum counts of 147 proteins identified, 25 proteins were upregulated and 49 were downregulated by DMSO treatment. CD11b/CD18 subunits of beta2-integrin Mac-1, CD35, and GPI-80, which are known to be upregulated during differentiation, were dominantly detected in the lipid rafts of DMSO-treated cells. Many known membrane proteins, G proteins, and cytoskeletal proteins were also detected and they showed characteristic distributions. Absolute quantification of nine proteins in the lipid rafts using internal standard peptides labeled with stable isotopes showed that the amount of protein almost corresponded to the results obtained by spectrum count. Identified proteins, expression of which was altered by DMSO treatment, are expected to be candidate proteins involved in differentiation and functions of neutrophils.     

Expeditious prediction of post-mortem changes in frozen fish meat using three-dimensional fluorescence fingerprints

The present study was conducted to characterize fluorophores in the fish body using three-dimensional fluorescence fingerprints (3D-FFs) and to utilize these 3D-FFs obtained from frozen horse mackerel (Trachurus japonicus) fillets to predict early post-mortem changes. Alive fish were sacrificed instantly, preserved in ice until 2 days, and then filleted, vacuum packed, and frozen. Subsequently, 3D-FFs of the frozen fillets were acquired using F-7000 aided with a fiber probe. Post-mortem freshness changes were tracked by measuring adenylate energy charge (AEC) values and nicotinamide adenine dinucleotide (NAD and NADH) content. Partial least squares regression models for predicting AEC values and NADH content in frozen fish meat showed good fittings, with R² of 0.90 and 0.85, by utilizing eight and five excitation wavelengths, respectively, based on their fluorescence features acquired from standard fluorophores. This novel approach of 3D-FFs could be utilized as an efficient technique for at-line monitoring of frozen fish quality.     

Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency

Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol.Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD(+) ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.     

Vessel Wall Inflammation of Takayasu Arteritis Detected by Contrast-Enhanced Magnetic Resonance Imaging: Association with Disease Distribution and Activity

Aims

The assessment of the distribution and activity of vessel wall inflammation is clinically important in patients with Takayasu arteritis. Magnetic resonance imaging (MRI) is a useful tool, but the clinical utility of late gadolinium enhancement (LGE) in Takayasu arteritis has yet to be determined. The aim of the present study was to evaluate the utility of LGE in assessing vessel wall inflammation and disease activity in Takayasu arteritis.

Methods and Results

We enrolled 49 patients with Takayasu arteritis who had undergone 1.5 T MRI. Patients were divided into Active (n = 19) and Inactive disease (n = 30) groups. The distribution of vessel wall inflammation using angiography and LGE was assessed by qualitative analysis. In 79% and 63% of patients in Active and Inactive groups, respectively, greater distribution of vessel wall inflammation was observed with LGE than with conventional angiography. MRI values of pre- and post-contrast signal-to-noise ratios (SNR), SNR increment (post-SNR minus pre-SNR), pre- and post-contrast contrast-to-noise ratios (CNR), and CNR increment (post-CNR minus pre-CNR) were evaluated at arterial wall sites with the highest signal intensity using quantitative analysis of post-contrast LGE images. No statistically significant differences in MRI parameters were observed between Active and Inactive groups. Contrast-enhanced MRI was unable to accurately detect active disease.

Conclusion

Contrast-enhanced MRI has utility in detecting the distribution of vessel wall inflammation but has less utility in assessing disease activity in Takayasu arteritis.     

Production of interleukin 3 and gamma-interferon by an antigen-specific mouse suppressor T cell clone

An interleukin 2 (IL 2)-dependent, keyhole limpet hemocyanin (KLH)-specific, mouse suppressor T cell clone, 3D10, was found to produce interleukin 3 (IL 3) and gamma-interferon (IFN-gamma) in response to T cell mitogens Con A and PHA. Different from KLH-specific suppressor factor (TsF) that was spontaneously released into the medium when cultured in IL 2-containing conditioned medium, the production of IL 3 and IFN-gamma was induced by mitogenic stimuli. IL 3, IFN-gamma, and TsF were separable by gel filtration through a Sephadex G-100 column, being recovered in fractions of m.w. 25,000 to 30,000, 45,000 to 50,000 and 60,000 to 70,000, respectively. On the other hand, minimum size of IL 3 and IFN-gamma were shown to be about 25,000 and 20,000, respectively, by determining the lymphokine activities contained in the extracts from slices of SDS gels. These results indicate that IFN-gamma was present as a homodimer or hetero-complex with another carrier protein(s), whereas IL 3 was present as a monomeric form. A highly positive correlation (a correlation coefficient r = 0.96) between the titers of IL 3 and IFN-gamma produced by seven subclones derived from 3D10 was obtained, suggesting that IL 3 and IFN-gamma are induced by a process with a common mechanism. 3D10 also produced IL 3 and IFN-gamma when cultured with its specific antigen, KLH, in the presence of antigen-presenting cells. When Con A-stimulated 3D10 cells were labeled with L-[35S]methionine, we found that at least three proteins, with m.w. of 35,000, 25,000, and 20,000, were specifically released into medium by the stimulation. The latter two may be IL 3 and IFN-gamma described above, respectively, because of the similarities in m.w.     

Leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B

Using synthetic leumorphin, we obtained antisera for leumorphin and set up two radioimmunoassays (RIAs) with different specificities. Gel exclusion chromatography coupled with the two RIAs showed the existence of a considerable amount of leumorphin-like peptide in water extracts from porcine neurointermediate pituitaries. Reverse phase high performance liquid chromatography revealed that leumorphin-like peptide in the water extracts was indistinguishable from synthetic leumorphin. These results along with potent opioid activity of leumorphin indicate that leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B.     

Homopoietic histocompatibility (Hh-1) phenotype and the regulation of its expression

Hybrid resistance (HR) is primarily controlled by the genes of the Hemopoietic histocompatibility-1 (Hh-1) locus within the H-2 complex. HR is a consequence of the Hh-1-controlled target determinants in homozygous parental strain mice and their absence in heterozygous F₁ hybrid mice. To examine the mechanism that controls the Hh-1 phenotype, three independent clones of somatic cell hybrids between parental lines EL-4 (C57BL/6 origin, H-2 ^b ) and R1 (C58 origin, H-2 ^k ) were studied. The line EL-4 is Hh-1^b-positive and is subject to HR by H-2 ^b heterozygous F₁ mice, but R1 lacks the Hh-1 ^b allele and is not susceptible to HR. Of the three hybrid clones, F263.2 is Hh-1^b-positive, whereas the other two, F262.2 and F264.2, are Hh-1-negative, as judged by these cells' capacity to compete in vivo with the grafted parental C57BL/6 bone marrow cells in the resistant (C57BL/6 × C3H)F₁ mice. All three clones express the H-2^b and H-2^k class I antigens equally well, are susceptible to activated NK cells to the same extent, and all carry four copies of chromosome 17. However, Southern analysis reveals that clone F263.2 contains three copies of H-2 ^b chromosome and one H-2 ^k , whereas the other two clones carry two copies each of the parental chromosome 17. The results suggest that the relative copy number of specific alleles is the crucial determinanr of the Hh-1 phenotype, and render unlikely both the gene dosage hypothesis and the trans-acting dominant suppression hypothesis to account for the noncodominant expression of the Hh-1 phenotype.