Search for MHC-associated genes in human: five new genes centromeric to HLA-DP with yet unknown functions

Taking advantage of five mouse genomic or cDNA probes [KE5(probe 14), KE4 (probe11), KE3 (probe7), KE2 (probe5), and SET] mapped on the H-2K region in mouse, we have identified and localized homologues of these five genes in the human major histocompatibility complex region (HKE5, HKE4, HKE3, HKE2, and HSET, respectively). Cosmid cloning and pulsed field gel electrophoresis analyses indicated that a human homologous gene, HKE5, is located 10 kilobases (kb) centromeric of the 2(XI) collagen (COL11A2) gene followed by HKE4. HKE3, closely linked to HKE2, is located 170 kb centromeric of HKE4. Furthermore, HSET is located 50 kb centromeric of HKE2. This gene organization outside the DP subregion is completely identical to that of the mouse H-2K region centromeric of I-Pb 3, a mouse homologue of the DPB gene, except the lack of genes corresponding to the H-2K and -K2 genes in human.     

A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels

Calpastatin, an endogenous inhibitor of calpain, is composed of domain L and four repetitive homologous domains 1-4. Domains 1-4 inhibit calpain, whereas domain L partially reprimes L-type Ca2+ channels for voltage-gated activation. In the present study, the effects on Ca2+ channel activity of four isoforms and a series of fragments of calpastatin domain L were investigated in guinea-pig ventricular myocytes with the patch-clamp method. With one exception, all the isoforms and fragment peptides that contained amino acid residues 54-64 of domain L reprimed the Ca2+ channels to comparable levels (9-15% of control activity) to those observed previously with a full-length form of calpastatin. These results suggest that the region containing amino acid residues 54-64 (EGKPKEHTEPK) is responsible for the Ca2+ channel repriming function of calpastatin domain L.     

Defective relocalization of ALS2/alsin missense mutants to Rac1-induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation

Loss of ALS2/alsin function accounts for several recessive motor neuron diseases. ALS2 is a Rab5 activator and its endosomal localization is regulated by Rac1 via macropinocytosis. Here, we show that the pathogenic missense ALS2 mutants fail to be localized to Rac1-induced macropinosomes as well as endosomes, which leads to loss of the ALS2 function as a Rab5 activator on endosomes. Further, these mutants lose the competence to enhance the formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes. Thus, Rac1-induced relocalization of ALS2 might be crucial to exert the ALS2 function associated with the autophagy-endolysosomal degradative pathway.     

Oxysterol‐binding protein recruitment and activity at the endoplasmic reticulum‐Golgi interface are independent of Sac1

Oxysterol‐binding protein (OSBP) localizes to endoplasmic reticulum (ER)‐Golgi contact sites where it transports cholesterol and phosphatidylinositol 4‐phosphate (PI‐4P), and activates lipid transport and biosynthetic activities. The PI‐4P phosphatase Sac1 cycles between the ER and Golgi apparatus where it potentially regulates OSBP activity. Here we examined whether the ER‐Golgi distribution of endogenous or ectopically expressed Sac1 influences OSBP activity. OSBP and Sac1 co‐localized at apparent ER‐Golgi contact sites in response to 25‐hydroxycholesterol (25OH), cholesterol depletion and p38 MAPK inhibitors. A Sac1 mutant that is unable to exit the ER did not localize with OSBP, suggesting that sterol perturbations cause Sac1 transport to the Golgi apparatus. Ectopic expression of Sac1 in the ER or Golgi apparatus, or Sac1 silencing, did not affect OSBP localization to ER‐Golgi contact sites, OSBP‐dependent activation of sphingomyelin synthesis, or cholesterol esterification in the ER. p38 MAPK inhibition and retention of Sac1 in the Golgi apparatus also caused OSBP phosphorylation and OSBP‐dependent activation of sphingomyelin synthesis at ER‐Golgi contacts. These results demonstrate that Sac1 expression in either the ER or Golgi apparatus has a minimal impact on the PI‐4P that regulates OSBP activity or recruitment to contact sites.      

Structural evidence for two isozymic forms and the carbohydrate attachment site of human gastric cathepsin E

The amino acid sequences in the NH2-terminal region and some other parts of human gastric cathepsin E were investigated. The NH2-terminal sequencing revealed that the cathepsin E preparation which had been activated at pH 4.0 contained one major and one minor isozymes in an approximate molar ratio of 3:1. The NH2-terminal sequence of the former was very similar to but partly different from that predicted from cDNA sequencing by Azuma et al., whereas the latter had an NH2-terminal sequence identical with the predicted sequence. These results provide structural evidence for the presence of at least two isozymic forms in human gastric cathepsin E. In addition, the site of carbohydrate attachment was elucidated by isolation and analysis of a glycopeptide fraction from an enzymatic digest of cathepsin E. A single carbohydrate chain was deduced to be attached to the asparagine residue at position 34 in the major isozyme and to the corresponding asparagine residue in the minor isozyme.     

Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt

Photosynthetic, nitrogen-fixing Anabaena strains play an important role in the carbon and nitrogen cycles in tropical paddy fields although they are salt sensitive. Improvement in salt tolerance of Anabaena cells by expressing glycine betaine–synthesizing genes is an interesting subject. Due to the absence of choline in cyanobacteria, choline-oxidizing enzyme could not be used for the synthesis of glycine betaine. Here, the genes encoding glycine-sarcosine and dimethylglycine methyltransferases (ApGSMT-DMT) from a halotolerant cyanobacterium Aphanothece halophytica were expressed in Anabaena sp. strain PCC7120. The ApGSMT-DMT-expressing Anabaena cells were capable of synthesizing glycine betaine without the addition of any substance. The accumulation level of glycine betaine in Anabaena increased with rise of salt concentration. The transformed cells exhibited an improved growth and more tolerance to salinity than the control cells. The present work provides a prospect to engineer a nitrogen-fixing cyanobacterium having enhanced tolerance to stress by manipulating de novo synthesis of glycine betaine.     

Enhanced Protection Against Peroxidation-Induced Mortality of Aortic Endothelial Cells by Ascorbic Acid-2-O-Phosphate Abundantly Accumulated in the Cell as the Dephosphorylated Form

Bovine aortic endothelial BAE-2 cells exposed to the peroxidizing agent, tert-butylhydroperoxide (t-BuOOH) or 2,4-nonadienal (NDE), suffered from disruption of cell membrane integrity and from reduction of mitochondrial dehydrogenase activity as assessed by fluorometry using ethidium homodimer and photometry using WST-1, respectively. The cells were protected from t-BuOOH-induced injury more markedly by L-ascorbic acid-2-O-phosphate (Asc2P) stably masked at the 2,3-enediol moiety, which is responsible for the antioxidant ability of L-ascorbic acid (Asc), than by Asc itself. In contrast, NDE-induced membrane disruption but not mitochondrial dysfunction was prevented by Asc2P, whereas Asc exhibited no prevention against both types of injury. The amount of intracellular Asc was 7.2- to 9.0-fold larger in Asc2P-administered BAE-2 cells, where the intact form Asc2P was not detected, than in Asc-administered cells as assessed by HPLC of cell extract with detection by coulometric ECD and W. During transmembrane influx into the cell, Asc2P was concentrated as highly as 70- to 90-fold relative to the extracellular Asc2P concentration, whereas Asc was 8-to 13-fold concentrated as estimated based on an intracellular water content of 0.59 pL/cell determined by [¹⁴C]PEG/gas chromatography. Thus, Asc2P but not Asc is highly concentrated in the aqueous phase of the cell after prompt dephosphorylation, and may thereby render the cell more resistant to t-BuOOH-peroxidation assumedly via scavenging of intracellular reactive oxygen species than to peroxidation with the less hydroplulic agent NDE.     

Sucrose-diet feeding induces gene expression of heat shock protein in rat brain under stress

Stress-induced hyperphagia is enhanced in the presence of sweets, particularly sucrose, which may act to attenuate stress. Recently, it was also reported that heat shock protein (HSP) may be involved in the defense against stress. To explore whether sucrose alters gene expression of HSP under stress, we determined the HSP mRNA levels in the hypothalamus, cerebellum, and cerebral cortex after restraint stress in sucrose-diet-fed rats. Competitive RT-PCR revealed that gene expressions of HSP27 in the cerebral cortex and cerebellum and of HSP70 in the cerebral cortex, hypothalamus, and cerebellum were induced by restraint stress under a sucrose-diet-fed condition. However, restraint stress by itself or sucrose diet alone did not induce expression of HSP27 or HSP70 mRNA in any of the three anatomical parts. It is suggested that sucrose facilitates the gene expression of HSP27 and HSP70 in brain after restraint stress, which may attenuate stress.     

Differential inactivation of seed exudate stimulation of Pythium ultimum sporangium germination by Enterobacter cloacae influences biological control efficacy on different plant species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.     

Failure to respond to interferon-α 2a therapy is associated with increased hepatic iron levels in patients with chronic hepatitis C

Recent reports suggest the hepatic iron concentration (HIC) may influence the activity of hepatitis and the response to interferon (IFN) therapy in patients with chronic hepatitis C (CH-C). We have evaluated iron status in 28 patients with CH-C and determined if pretreatment iron status can predict the response to IFN-α therapy in these patients. Increased serum iron, transferrin saturation, and ferritin levels were observed in 3 (11%), 11 (39%), and 5 (18%) patients, respectively. Hepatic iron deposits were histologically detected in 17 (61%) patients, and 14 of them had stainable hepatocytic iron. However, all HIC values were within the normal range (203–1279 μg/g). Seven of 17 patients treated with IFN-α for 6 mo had normalization of serum transaminases and disappearance of serum HCV-RNA (responders). Nonresponders had a significantly higher median HIC compared with responders (710 vs 343 μg/g, respectively;p < 0.05). There was no significant difference in other pretreatment iron parameters, serum HCV-RNA level, or HCV-genotype between responders and nonresponders. In conclusion, mild hepatic iron accumulation occurs in patients with CH-C. Increased hepatic iron stores are associated with poor response to IFN therapy. Pretreatment HIC may be an additional host-specific parameter with a predictive value for responsiveness to IFN therapy, in addition to well-known predictive viral factors.