Hypothalamic neuronal histamine modulates febrile response but not anorexia induced by lipopolysaccharide

This study examined the contribution of hypothalamic neuronal histamine (HA) to the anorectic and febrile responses induced by lipopolysaccharide (LPS), an exogenous pyrogen, and the endogenous pyrogens interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Intraperitoneal (ip) injection of LPS, IL-1beta, or TNF-alpha suppressed 24-hr cumulative food intake and increased rectal temperature in rats.To analyze the histaminergic contribution, rats were pretreated with intracerebroventricular (icv) injection of 2.44 mmol/kg or ip injection of 244 mmol/kg of alpha-fluoromethylhistidine (FMH), a suicide inhibitor of histidine decarboxylase (HDC), to deplete neural HA. The depletion of neural HA augmented the febrile response to ip injection of LPS and IL-1beta and alleviated the anorectic response to ip injection of IL-1beta. However, the depletion of neural HA did not modify the LPS-induced anorectic response or TNF-alpha-induced febrile and anorectic responses. Consistent with these results, the rate of hypothalamic HA turnover, assessed by the accumulation of tele-methylhistamine (t-MH), was elevated with ip injections of LPS and IL-1beta, but unaffected by TNF-alpha at equivalent doses. This suggests that (i) LPS and IL-1beta activate hypothalamic neural HA turnover; (ii) hypothalamic neural HA suppresses the LPS- and IL-1beta-induced febrile responses and accelerates the IL-1beta-induced anorectic response; and (iii) TNF-alpha modulates the febrile and anorectic responses via a neural HA-independent pathway. Therefore, hypothalamic neural HA is involved in the IL-1beta-dominant pathway, rather than the TNF-alpha-dominant pathway, preceding the systemic inflammatory response induced by exogenous pyrogens, such as LPS. Further research on this is needed.     

Class I and class II chitin synthases are involved in septum formation in the filamentous fungus Aspergillus nidulans

The class II and class I chitin synthases of the filamentous fungus Aspergillus nidulans are encoded by chsA and chsC, respectively. Previously, we presented several lines of evidence suggesting that ChsA and ChsC have overlapping functions in maintaining cell wall integrity. In order to determine the functions of these chitin synthases, we employed electron and fluorescence microscopy and investigated in detail the cell wall of a DeltachsA DeltachsC double mutant (DeltaAC mutant) along with the localization of ChsA and ChsC. In the lateral cell wall of the DeltaAC mutant, electron-transparent regions were thickened. Septa of the DeltaAC mutant were aberrantly thick and had a large pore. Some septa were located abnormally close to adjacent septa. A functional hemagglutinin (HA)-tagged ChsA (HA-ChsA) and a functional FLAG-tagged ChsC (FLAG-ChsC) were each localized to a subset of septation sites. Comparison with the localization pattern of actin, which is known to localize at forming septa, suggested that ChsA and ChsC transiently exist at the septation sites during and shortly after septum formation. Double staining of HA-ChsA and FLAG-ChsC indicated that their localizations were not identical but partly overlapped at the septation sites. Fluorescence of FLAG-ChsC, but not of HA-ChsA, was also observed at hyphal tips. These data indicate that ChsA and ChsC share overlapping roles in septum formation.     

Evaluation of three methods for effective extraction of DNA from human hair

In this paper we evaluate three different methods for extracting DNA from human hair i.e. the Chelex method, the QIAamp DNA Mini Kit method and the ISOHAIR method. Analysis of DNA prepared from dyed hairs with the ISOHAIR method suggested that the DNA extracts contained PCR inhibitors. On the other hand, few inhibition was observed when DNA from dyed hairs were extracted using the Chelex method and the QIAamp DNA Mini Kit method. In conclusion, the Chelex method is recommended for PCR experiments in view of its simplicity and cost-effectiveness. To assess the reliability of the Chelex method for the extraction of genomic DNA from both natural and dyed hair samples, minisatellite variant repeat (MVR)-polymerase chain reaction (PCR) patterns of Chelex-extracted DNA were compared using hairs (three natural black hairs and three dyed hairs) with buccal swabs from six individuals. Complete agreement was observed between hair and swab samples in each individual, proving the utility of the Chelex method.     

Platelet-derived growth factor-BB (PDGF-BB) induces differentiation of bone marrow endothelial progenitor cell-derived cell line TR-BME2 into mural cells, and changes the phenotype

Blood vessels are composed of endothelial cells (EC) and mural cells, and the interaction between EC and mural cells is essential for the development and maintenance of the vasculature. EC differentiate from bone marrow-derived endothelial progenitor cells (EPC). Recently, we established a conditionally immortalized bone marrow EPC-derived cell line, TR-BME2, and a brain capillary EC (BCEC) line, TR-BBB, from temperature-sensitive-SV40 T-antigen gene transgenic rats. To understand the function of EPC, it is important to analyze the difference between EPC and mature EC such as BCEC. In this study, we identified EPC-specific genes by means of subtractive hybridization between TR-BME2 and TR-BBB. There was no significant difference between TR-BME2 and TR-BBB in the mRNA level of annexin II, which is expressed in EC. In contrast, the mRNA level of smooth muscle cell (SMC) markers such as smooth muscle protein 22 (SM22), calvasculin, and platelet-derived growth factor (PDGF) receptor-beta, was higher in TR-BME2 than in TR-BBB. Moreover, the mRNA level of contractile SMC markers, such as smooth muscle alpha-actin and SM22, was increased in the absence of EC growth factors, such as vascular endothelial growth factor. The mRNA level of synthetic SMC markers, such as matrix Gla protein, was increased by the addition of PDGF-BB. The SMC derived from TR-BME2 showed an altered phenotype, from contractile-type to synthetic-type, when they were cultured in the absence of PDGF-BB. These results show that TR-BME2 cells have higher levels of SMC markers compared with mature EC, and can differentiate into contractile- or synthetic-type SMC.     

Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant

A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.     

A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor

Ligand-stimulated growth factor receptors are rapidly internalized and transported to early endosomes. Unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to the same organelle. At early endosomes, stimulated receptors are sorted for the lysosomal degradation pathway, whereas unstimulated receptors are mostly recycled back to the cell surface. To investigate the role of Hrs, an early endosomal protein, in this sorting process, we overexpressed Hrs in HeLa cells and examined the intracellular trafficking of epidermal growth factor receptor (EGFR) in EGF-stimulated and unstimulated cells. Overexpression of Hrs inhibited the trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. On the other hand, overexpression of Hrs mutants with a deletion or a point mutation within the FYVE domain did not inhibit the trafficking. These results suggest that Hrs regulates the sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to reside in a microdomain of early endosomes, plays an essential role in the function of Hrs.     

Copper-mediated LDL oxidation by homocysteine and related compounds depends largely on copper ligation

Oxidation of low-density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Elevated plasma homocysteine is an accepted risk factor for atherosclerosis, and may act through LDL oxidation, although this is controversial. In this study, homocysteine at physiological concentrations is shown to act as a pro-oxidant for three stages of copper-mediated LDL oxidation (initiation, conjugated diene formation and aldehyde formation), whereas at high concentration, it acts as an antioxidant. The affinity for copper of homocysteine and related copper ligands homocysteine, cystathionine and djenkolate was measured, showing that at high concentrations (100 microM) under our assay conditions, they bind essentially all of the copper present. This is used to rationalise the behaviour of these ligands, which stimulate LDL oxidation at low concentration but generally inhibit it at high concentration. Albumin strongly reduced the effect of homocystine on lag time for LDL oxidation, suggesting that the effects of homocystine are due to copper binding. In contrast, copper binding does not fully explain the pro-oxidant behaviour of low concentrations of homocysteine towards LDL, which appears in part at least to be due to stimulation of free radical production. The likely role of homocysteine in LDL oxidation in vivo is discussed in the light of these results.     

Structural and functional relationship among diamines in terms of inhibition of cell growth

Following the report that agmatine has an anti-proliferative effect on cell growth through induction of antizyme [Satriano et al. (1998) J. Biol. Chem. 273, 15313-15316], we examined the effects of 16 different diamines on cell growth. Many diamines had little or no effect on cell growth, but agmatine and 1,6-hexanediamine had anti-proliferative effects, with agmatine having the strongest effect. Inhibition of cell growth occurred after 2 days, and inhibitory effects paralleled the degree of antizyme induction. Decreased spermine levels indicated that induction of spermidine/spermine N(1)-acetyltransferase was also involved in the inhibition of cell growth by agmatine and 1,6-hexanediamine. The frameshift efficiency (ratio of antizyme synthesis with or without frameshift) measured in a rabbit reticulocyte cell-free system was also increased by 1,3-propanediamine and cis-1,4-cyclohexanediamine in addition to agmatine and 1,6-hexanediamine. However, the intracellular levels of 1,3-propanediamine and cis-1,4-cyclohexanediamine were low when these compounds were added to the cell-culture medium. Other diamines had no effect on cell growth or frameshift efficiency. The results suggest that the presence of two amino-groups separated by an appropriate distance is important for the enhancement of frameshifting by diamines.