Further characterization of high mobility group box 1 (HMGB1) as a proinflammatory cytokine: central nervous system effects

High mobility group box 1 (HMGB1), an abundant, highly conserved cellular protein, is widely known as a nuclear DNA-binding protein. HMGB1 has been recently implicated as a proinflammatory cytokine because of its role as a late mediator of endotoxin lethality and ability to stimulate release of proinflammatory cytokines from monocytes. Production of central cytokines is a critical step in the pathway by which endotoxin and peripheral proinflammatory cytokines, including interleukin-1beta (IL-1) and tumor necrosis factor-alpha (TNF), produce sickness behaviors and fever. Intracerebroventricular (ICV) administration of HMGB1 has been shown to increase TNF expression in mouse brain and induce aphagia and taste aversion. Here we show that ICV injections of HMGB1 induce fever and hypothalamic IL-1 in rats. Furthermore, we show that intrathecal administration of HMGB1 produces mechanical allodynia (lowering of the response threshold to calibrated stimuli). Finally, while endotoxin (lipopolysaccharide, LPS) administration elevates IL-1 and TNF mRNA in various brain regions, HMGB1 mRNA is unchanged. It remains possible that HMGB1 protein is released in brain in response to LPS. Nonetheless, these data suggest that HMGB1 may play a role as an endogenous pyrogen and support the concept that HMGB1 has proinflammatory characteristics within the central nervous system.     

Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.     

Pilot study of elevated levels of insulin-like growth factor-binding protein-2 as indicators of hepatocellular carcinoma

BACKGROUND/AIMS: Insulin-like growth factor-binding protein-2 (IGFBP-2) is expressed in many malignant tissues, and elevated serum levels can be indicators of tumour activity in addition to conventional tumour markers. Our aim was to evaluate the role of IGFBP-2 levels together with insulin-like growth factor (IGF)-I, IGF-II and IGFBP-3 in the diagnostic work-up of patients with hepatocellular carcinoma (HCC). METHODS: In 50 (39 males, 11 females) histologically confirmed and TNM-graded patients with HCC who had not received adjuvant chemotherapy, the basal serum levels of IGF-I, IGF-II, IGFBP-3, IGFBP-2 and alpha-fetoprotein (AFP) were measured. The median age of the patients was 66 (37-84) years, body mass index was normal (25 (35-16) kg/m2). RESULTS: The levels of IGF-I, IGF-II and IGFBP-3 were diminished, as is the case when nutrition, hepatic function and growth hormone (GH) secretion are decreased. The levels of AFP and IGFBP-2 were markedly high. In 37 cases, IGFBP-2 levels were above the age-related norm, and in 40 cases AFP levels were also elevated. In 3 cases, both AFP and IGFBP-3 were normal, and in 4 cases AFP was high but IGFBP-2 normal, whereas in 10 cases AFP was normal but IGFBP-2 was high. CONCLUSIONS: In addition to AFP, IGFBP-2 appears to be a suitable marker for the evaluation of the serological status of HCC patients. A longitudinal study during disease management is required to assess the full potential of IGFBP-2 measurements as a marker.     

The parasitome of the phytonematode Heterodera glycines

Parasitism genes expressed in the esophageal gland cells of phytonematodes encode secretions that control the complex process of plant parasitism. In the soybean cyst nematode, Heterodera glycines, the parasitome, i.e., the secreted products of parasitism genes, facilitate nematode migration in soybean roots and mediate the modification of root cells into elaborate feeding cells required to support the growth and development of the nematode. With very few exceptions, the identities of these secretions are unknown, and the mechanisms of cyst nematode parasitism, therefore, remain obscure. The most direct and efficient approach for cloning parasitism genes and rapidly advancing our understanding of the molecular interactions during nematode parasitism of plants is to create gland cell-specific cDNA libraries using cytoplasm microaspirated from the esophageal gland cells of various parasitic stages. By combining expressed sequence tag analysis of a gland cell cDNA library with high throughput in situ expression localization of clones encoding secretory proteins, we obtained the first comprehensive parasitome profile for a parasitic nematode. We identified 51 new H. glycines gland-expressed candidate parasitism genes, of which 38 genes constitute completely novel sequences. Individual parasitome members showed distinct gland cell expression patterns throughout the parasitic cycle. The parasitome complexity discovered paints a more elaborate picture of host cellular events under specific control by the nematode parasite than previously hypothesized.     

Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway

A chromosomal fragment has been identified in a gene bank from Escherichia coli, which augmented the yield of cysteine in an industrial production strain. Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids (Orf299) was responsible. Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein. Mutational interruption of orf299 did not cause a distinct phenotype; however, transformants overexpressing orf299 had lost the ability to grow in minimal medium unless it was supplemented with a source of reduced sulphur compounds, and they excreted considerable amounts of cysteine and O-acetyl-L-serine, especially in the presence of thiosulphate. Most of the cysteine was found to be masked in 2-methyl-2,4-thiazolidinedicarboxylic acid. N-acetyl-L-serine was also present in the medium, but it is open to question whether it represents a primary excretion product. Measurement of the induction status of the cysteine regulon by means of a cysK'-'lacZ gene fusion demonstrated that the regulon is not induced upon growth in the presence of a poor sulphur source and that the introduction of a constitutive cysB allele alleviates this deficiency. The results indicate that orf299 codes for an export pump for different metabolites of the cysteine pathway. Its relation to other efflux systems and the physiological role are discussed.     

Characterization of the NifU and NifS Fe-S cluster formation proteins essential for viability in Helicobacter pylori

The Fe-S cluster formation proteins NifU and NifS are essential for viability in the ulcer causing human pathogen Helicobacter pylori. Obtaining viable H. pylori mutants upon mutagenesis of the genes encoding NifU and NifS was unsuccessful even by growing the potential transformants under many different conditions including low O(2) atmosphere and supplementation with both ferric and ferrous iron. When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, this second copy of the gene could be disrupted at high frequency. This indicates that the procedures used for transformation were capable of nifU mutagenesis, so that the failure to recover mutants is solely due to the requirement of nifU for H. pylori viability. H. pylori NifU and NifS were expressed in Escherichia coli and purified to near homogeneity, and the proteins were characterized. Purified NifU is a red protein that contains approximately 1.5 atoms of iron per monomer. This iron was determined to be in the form of a redox-active [2Fe-2S](2+,+) cluster by characteristic UV-visible, EPR, and MCD spectra. The primary structure of NifU also contains the three conserved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe-S cluster for insertion into apoprotein. Purified NifS has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate containing enzyme. NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from L-cysteine, in agreement with its proposed role as a sulfur donor to Fe-S clusters. The results here indicate that the NifU type of Fe-S cluster formation proteins is not specific for maturation of the nitrogenase proteins and, as H. pylori lacks other Fe-S cluster assembly proteins, that the H. pylori NifS and NifU are responsible for the assembly of many (non-nitrogenase) Fe-S clusters.     

Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga

We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH(2)-terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non-photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH-terminal extension, which shows high homology exclusively to the COOH-termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron-containing holorubredoxin and zinc-substituted protein. Preliminary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd-type iron site. One-dimensional nuclear magnetic resonance spectroscopy of recombinant Zn-rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.