Essential roles of high-mobility group box 1 in the development of murine colitis and colitis-associated cancer

High-mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as a proinflammatory cytokine. We measured the HMGB1 concentration in the sera of mice with chemically induced colitis (DSS; dextran sulfate sodium salt) and found a marked increase. Inhibition of HMGB1 by neutralizing anti-HMGB1 antibody resulted in reduced inflammation in DSS-treated colons. In macrophages, HMGB1 induces several proinflammatory cytokines, such as IL-6, which are regulated by NF-kappaB activation. Two putative sources of HMGB1 were explored: in one, bacterial factors induce HMGB1 secretion from macrophages and in the other, necrotic epithelial cells directly release HMGB1. LPS induced a small amount of HMGB1 in macrophages, but macrophages incubated with supernatant prepared from necrotic cells and containing large amounts of HMGB1 activated NF-kappaB and induced IL-6. Using the colitis-associated cancer model, we demonstrated that neutralizing anti-HMGB1 antibody decreases tumor incidence and size. These observations suggest that HMGB1 is a potentially useful target for IBD treatment and the prevention of colitis-associated cancer.     

Molecular cloning and expression of human neurochondrin-1 and -2.

Human neurochondrins have been cloned from a brain cDNA library. The human neurochondrin-1 and -2 predict leucine-rich (15.8 and 15.9%) proteins of 729 and 712 amino acid residues, with molecular weights of 78.9 and 77.2 kDa, respectively. The deduced amino acid sequence indicates 98% identity among human, mouse and rat species. Northern analysis indicates that about 4 kb human neurochondrin mRNAs are abundant in the fetal and the adult brain.     

Frequencies in the Japanese Population of HFE Gene Mutations

We studied the frequencies of C282Y and H63Dmutations in the HFE gene, thought to be responsible forhereditary hemochromatosis (HH), in 504 chromosomesobtained from 252 unrelated Japanese. Allele-specific PCR and PCRrestriction fragment lengthpolymorphism methods revealed that the C282Y mutationwas not found and the H63D mutation was low in frequency(at only 0.99%) compared with data from European people. Since most HH is thought to be associated withthe HFE gene mutation, the low incidence of thesemutations is a likely reason for the rarity of thisdisease in the Japanese population.     

NIMA-related kinases: isolation and characterization of murine nek3 and nek4 cDNAs, and chromosomal localization of nek1, nek2 and nek3.

The Aspergillus NIMA kinase plays a key role in controlling entrance into mitosis, and recent evidence suggests that mammalian NIMA-related kinases perform similar functions. We report here the cloning of the mouse nek3 and nek4 genes. Mouse nek3 is probably the ortholog of the partially sequenced, human nek3, whereas murine nek4 cDNA is probably the ortholog of human STK2. Nek4 is highly conserved between mouse and human, whereas Nek3 is somewhat less conserved (96.5 and 88% identity in the kinase domains, respectively). Northern analysis shows preferential expression of nek3 in mitotically active tissue, whereas nek4 is highly abundant in the testis. Within the developing testicular germ cells, in-situ analysis demonstrated that nek1, 2 and 4 exhibit differential patterns of expression, suggesting overlapping, but non-identical functions. Linkage analysis, using the mouse recombinant inbred strain panel (BXD), was used to localize nek1, 2 and 3. nek1 was mapped between Cpe and D8Mit8 on chromosome 8 at around 32cM, nek2 was mapped to the distal region of chromosome 1, and nek3 was mapped to the most centromeric region of chromosome 8.     

The Biogeochemistry of Carbon at Hubbard Brook

The biogeochemical behavior of carbon in the forested watersheds of the Hubbard Brook Experimental Forest (HBEF) was analyzed in long-term studies. The largest pools of C in the reference watershed (W6) reside in mineral soil organic matter (43% of total ecosystem C) and living biomass (40.5%), with the remainder in surface detritus (14.5%). Repeated sampling indicated that none of these pools was changing significantly in the late-1990s, although high spatial variability precluded the detection of small changes in the soil organic matter pools, which are large; hence, net ecosystem productivity (NEP) in this 2nd growth forest was near zero (± about 20 g C/m²-yr) and probably similar in magnitude to fluvial export of organic C. Aboveground net primary productivity (ANPP) of the forest declined by 24% between the late-1950s (462 g C/m²-yr) and the late-1990s (354 g C/m²-yr), illustrating age-related decline in forest NPP, effects of multiple stresses and unusual tree mortality, or both. Application of the simulation model PnET-II predicted 14% higher ANPP than was observed for 1996–1997, probably reflecting some unknown stresses. Fine litterfall flux (171 g C/m²-yr) has not changed much since the late-1960s. Because of high annual variation, C flux in woody litterfall (including tree mortality) was not tightly constrained but averaged about 90 g C/m²-yr. Carbon flux to soil organic matter in root turnover (128 g C/m²-yr) was only about half as large as aboveground detritus. Balancing the soil C budget requires that large amounts of C (80 g C/m²-yr) were transported from roots to rhizosphere carbon flux. Total soil respiration (TSR) ranged from 540 to 800 g C/m²-yr across eight stands and decreased with increasing elevation within the northern hardwood forest near W6. The watershed-wide TSR was estimated as 660 g C/m²-yr. Empirical measurements indicated that 58% of TSR occurred in the surface organic horizons and that root respiration comprised about 40% of TSR, most of the rest being microbial. Carbon flux directly associated with other heterotrophs in the HBEF was minor; for example, we estimated respiration of soil microarthropods, rodents, birds and moose at about 3, 5, 1 and 0.8 g C/m²-yr, respectively, or in total less than 2% of NPP. Hence, the effects of other heterotrophs on C flux were primarily indirect, with the exception of occasional irruptions of folivorous insects. Hydrologic fluxes of C were significant in the watershed C budget, especially in comparison with NEP. Although atmospheric inputs (1.7 g C/m²-yr) and streamflow outputs (2.7 g C/m²-yr) were small, larger quantities of C were transported within the ecosystem and a more substantial fraction of dissolved C was transported from the soil as inorganic C and evaded from the stream as CO₂ (4.0 g C/m²-yr). Carbon pools and fluxes change rapidly in response to catastrophic disturbances such as forest harvest or major windthrow events. These changes are dominated by living vegetation and dead wood pools, including roots. If biomass removal does not accompany large-scale disturbance, the ecosystem is a large net source of C to the atmosphere (500–1200 g C/m²-yr) for about a decade following disturbance and becomes a net sink about 15–20 years after disturbance; it remains a net sink of about 200–300 g C/m²-yr for about 40 years before rapidly approaching steady state. Shifts in NPP and NEP associated with common small-scale or diffuse forest disturbances (e.g., forest declines, pathogen irruptions, ice storms) are brief and much less dramatic. Spatial and temporal patterns in C pools and fluxes in the mature forest at the HBEF reflect variation in environmental factors. Temperature and growing-season length undoubtedly constrain C fluxes at the HBEF; however, temperature effects on leaf respiration may largely offset the effects of growing season length on photosynthesis. Occasional severe droughts also affect C flux by reducing both photosynthesis and soil respiration. In younger stands nutrient availability strongly limits NPP, but the role of soil nutrient availability in limiting C flux in the mature forest is not known. A portion of the elevational variation of ANPP within the HBEF probably is associated with soil resource limitation; moreover, sites on more fertile soils exhibit 20–25% higher biomass and ANPP than the forest-wide average. Several prominent biotic influences on C pools and fluxes also are clear. Biomass and NPP of both the young and mature forest depend upon tree species composition as well as environment. Similarly, litter decay differs among tree species and forest types, and forest floor C accumulation is twice as great in the spruce–fir–birch forests at higher elevations than in the northern hardwood forests, partly because of inherently slow litter decay and partly because of cold temperatures. This contributes to spatial patterns in soil solution and streamwater dissolved organic carbon across the Hubbard Brook Valley. Wood decay varies markedly both among species and within species because of biochemical differences and probably differences in the decay fungi colonizing wood. Although C biogeochemistry at the HBEF is representative of mountainous terrain in the region, other sites will depart from the patterns described at the HBEF, due to differences in site history, especially agricultural use and fires during earlier logging periods. Our understanding of the C cycle in northern hardwood forests is most limited in the area of soil pool size changes, woody litter deposition and rhizosphere C flux processes.     

Application of positron emission tomography to neuroimaging in sports sciences

To investigate exercise-induced regional metabolic and perfusion changes in the human brain, various methods are available, such as positron emission tomography (PET), functional magnetic resonance imaging (fMRI), near-infrared spectroscopy (NIRS) and electroencephalography (EEG). In this paper, details of methods of metabolic measurement using PET, [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) and [¹⁵O]radio-labelled water ([¹⁵O]H₂O) will be explained.Functional neuroimaging in the field of neuroscience was started in the 1970s using an autoradiography technique on experimental animals. The first human functional neuroimaging exercise study was conducted in 1987 using a rough measurement system known as ¹³³Xe inhalation. Although the data was useful, more detailed and exact functional neuroimaging, especially with respect to spatial resolution, was achieved by positron emission tomography. Early studies measured the cerebral blood flow changes during exercise. Recently, PET was made more applicable to exercise physiology and psychology by the use of the tracer [¹⁸F]FDG. This technique allowed subjects to be scanned after an exercise task is completed but still obtain data from the exercise itself, which is similar to autoradiography studies.In this report, methodological information is provided with respect to the recommended protocol design, the selection of the scanning mode, how to evaluate the cerebral glucose metabolism and how to interpret the regional brain activity using voxel-by-voxel analysis and regions of interest techniques (ROI).Considering the important role of exercise in health promotion, further efforts in this line of research should be encouraged in order to better understand health behavior. Although the number of research papers is still limited, recent work has indicated that the [¹⁸F]FDG-PET technique is a useful tool to understand brain activity during exercise.     

A case of fibrosarcoma on the perivertebral surface of a ferret with hind limb paralysis

A 2.5 year-old female ferret had a stiff palpable mass arising from the dorsal surface of the thoracic (T) to lumbar (L) vertebrae with paralysis of the hind limbs. By myelography the dorsal and ventral lines of contrast were not observed in the area forwarded of L3. Grossly, the tumor encircled the dorsal vertebrae. Microscopically, tumor cells were proliferated intimately and were attached to the vertebrae surface involving surrounding fatty and connective tissues. The tumor consisted of fibroblastic cells with prominent cellular atypia. The bromodeoxyuridine (BrdU) labeling index to examine cellular kinetics was high (11.8%). Based on macro and micropathological features, the present tumor was diagnosed as periosteal fibrosarcoma arising from perivertebral connective tissue.     

Anti-fertilization activity of a spirocyclic sesquiterpene isocyanide isolated from the marine sponge Geodia exigua and related compounds

(-)-10-epi-Axisonitrile-3, a spirocyclic sesquiterpene isocyanide obtained from the marine sponge Geodia exigua, immobilized sperm of sea urchin and starfish to block fertilization at the minimum effective concentration of 0.4 microg/ml. On the other hand, fertilized eggs developed normally to the gastrula stage in the presence of a 250-times higher concentration of the isocyanide. Analysis by (31)P NMR revealed an accumulation of phosphocreatine and a depletion of inorganic phosphate in the isocyanide-treated sperm, suggesting that (-)-10-epi-axisonitrile-3 inhibited the phosphocreatine shuttle participating in the high-energy phosphate metabolism, thereby immobilizing sperm to block fertilization. No analogs of (-)-10-epi-axisonitrile-3 containing different functionalities or isocyanides with different carbon skeleton exhibited such activity.     

Subretinal delivery of adeno‐associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice

Background

Adeno‐associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long‐term therapy is the development of immune responses against the vector or transgene product.

Methods

We evaluated cellular and humoural responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression.

Results

Following subretinal administration of vector, splenocytes and T‐cells from draining lymph nodes showed minimal activation following stimulation by co‐culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low‐dose AAV.hRPE65.hRPE65 to both eyes of RPE65^?/? mice resulted in transgene expression and functional rescue, but re‐administration of high‐dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye.

Conclusions

These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose‐dependent. Low‐dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration. Copyright © 2009 John Wiley & Sons, Ltd.