An exfoliative toxin A-converting phage isolated from Staphylococcus aureus strain ZM

Exfoliative toxin A (ETA) causes staphylococcal scalded-skin syndrome in children. The gene for ETA was believed to be coded by the chromosomal DNA. We isolated temperate phages from an ETA-producing strain, ZM, using a restriction minus strain, 1039, as an indicator. One of the prophages, designated phi-ZM-1 mediated lysogenic conversion of ETA. The polymerase chain reaction assay of the eta gene revealed that phage phi-ZM-1 carries the structural gene for ETA.     

Cold denaturation of alpha-lactalbumin

We have investigated the thermal unfolding of bovine alpha-lactalbumin by means of circular dichroism spectroscopy in the far- and near-ultraviolet regions, and shown that the native alpha-lactalbumin undergoes heat and cold denaturation. The guanidine hydrochloride-induced unfolding of alpha-lactalbumin was also investigated by circular dichroism spectroscopy at various temperatures from 261 to 318 K. It is shown that the population of the molten globule state is strongly dependent on temperature and that the molten globule state does not accumulate during the guanidine hydrochloride-induced unfolding transition at 261 K. Our results indicate that the molten globule state of alpha-lactalbumin undergoes cold denaturation as the native alpha-lactalbumin does, and that the heat capacity change of unfolding from the molten globule to the unfolded state is positive and significant. The present results further support the idea that the molten globule and the unfolded states do not belong to the same thermodynamic state, and that the native, molten globule and unfolded states are sufficient for interpreting the guanidine hydrochloride-induced unfolding behavior of alpha-lactalbumin.     

Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system

A bacterial expression system for the variable region fragments (Fvs) of the anti-MUC1 tumor antigen antibody MUSE11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH-linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS)(3) or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC1 antibody MUSE11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the first report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH.     

Intracellular cytokine profile of T cells from children with acute lymphoblastic leukemia

Purpose: During an ongoing immune response, cytokines produced by T helper types 1 (Th1) and 2 (Th2) together with T cytotoxic types 1 (Tc1) and 2 (Tc2) are critical to the effectiveness of that response. Dysregulated expansion of one or the other subset may contribute to the impaired function of the T-cell-mediated immune system in cancer patients. In the present study we have investigated whether such dysregulation might exist in children with acute lymphoblastic leukemia (ALL). Methods: We analyzed 61 blood samples from 45 children with B cell precursor ALL and 16 healthy children. Interleukin(IL)-2, IL-4, and interferon γ (IFNγ) production of their respective purified CD4⁺ and CD8⁺ T cells were assessed at the single-cell level by intracellular-cytokine-staining flow cytometry. Results: At the time of diagnosis, IL-2-producing cell populations in CD4⁺ and CD8⁺ T cells were reduced below the normal range in 31 of 44 (70.5%) and 23 of 38 (60.5%) cases respectively. Similarly, IFNγ-producing cell populations in CD4⁺ and CD8⁺ T cells decreased in 17 of 44 (38.6%) and 18 of 38 (47.4%) cases respectively. Conversely cell populations capable of IL-4 production in CD4⁺ and CD8⁺ T cell subsets were increased in 13 of 30 (43.3%) and 15 of 30 (50.0%) cases respectively. Therefore, the Th1-to-Th2 and Tc1-to-Tc2 ratios (1.6 ± 2.2 and 7.7 ± 6.7 respectively) were significantly lower in peripheral blood T cells of ALL patients (n = 30) than those (6.0 ± 2.9 and 20.1 ± 10.3 respectively) in 15 healthy controls (P < 0.0001). Although both CD45RA⁺/CD4⁺ and CD45RA⁺/CD8⁺ cells significantly increased in 43 ALL patients (P < 0.05), there existed no apparent correlation between CD45 isoform expression and cytokine (IL-2 and IFNγ) production. Interestingly, the ability to produce both IL-2 and IFNγ was recovered in 8 cases examined, after complete remission had been achieved. Conclusion: These observations suggest that, in both CD4⁺ and CD8⁺ T cells of ALL patients, there is a dysregulation in the functionality of Th1 (Tc1) and Th2 (Tc2) cells with a gross reduction of Th1 (Tc1) cell populations and an expansion in Th2 (Tc2). Received: 12 November 1999 / Accepted: 2 January 2000     

The CD16- CD56(bright) NK cell subset is resistant to reactive oxygen species produced by activated granulocytes and has higher antioxidative capacity than the CD16+ CD56(dim) subset

Human NK cells can be divided into CD56(dim) and CD56(bright) subsets. These two types of NK cells respond to different types of stimuli, with CD56(dim) NK cells having direct cytotoxic ability and CD56(bright) NK cells having mainly an immunoregulatory function. We show that the CD16+ CD56(dim) NK subset is characterized by sensitivity to cell death induced by activated granulocytes. We identified hydrogen peroxide (H2O2) as the major effector molecule responsible for the cytotoxic effect of granulocytes on CD56(dim) NK cells, because the ability of granulocytes to kill CD56(dim) NK cells was completely abrogated in the presence of the hydrogen peroxide scavenger catalase. When exposing NK cells to H2O2, CD56(dim) cells showed rapid mitochondrial depolarization and down-regulation of activating NKRs, eventually resulting in cell death, whereas CD56(bright) cells remained unaffected. The difference in sensitivity to H2O2 was mirrored by a difference in intracellular oxidation levels between CD56(dim) and CD56(bright) NK cells, and cell lysates from the latter subset possessed a greater ability to block H2O2-mediated oxidation. Our data may explain the preferential accumulation of CD56(bright) NK cells often seen in environments rich in reactive oxygen species, such as at sites of chronic inflammation and in tumors.     

Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.     

Activation of extracellular signal-regulated protein kinases 5 in primary afferent neurons contributes to heat and cold hyperalgesia after inflammation

Heat and cold hyperalgesia is a common feature of inflammatory pain. To investigate whether activation of extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1, in primary sensory neurons participates in inflammatory pain, we examined the phosphorylation of ERK5 in the dorsal root ganglion (DRG) after peripheral inflammation. Inflammation induced by complete Freund's adjuvant produced heat and cold hyperalgesia on the ipsilateral hind paw and induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-expressing small- and medium-size neurons. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. ERK5 antisense, but not mismatch, oligodeoxynucleotide decreased the activation of ERK5 and suppressed inflammation-induced heat and cold hyperalgesia. Furthermore, the inhibition of ERK5 blocked the induction of transient receptor potential channel TRPV1 and TRPA1 expression in DRG neurons after peripheral inflammation. Our results show that ERK5 activated in DRG neurons contribute to the development of inflammatory pain. Thus, blocking ERK5 signaling in sensory neurons that has the potential for preventing pain after inflammation.     

Molecular properties of rhodopsin and rod function

Signal transduction in rod cells begins with photon absorption by rhodopsin and leads to the generation of an electrical response. The response profile is determined by the molecular properties of the phototransduction components. To examine how the molecular properties of rhodopsin correlate with the rod-response profile, we have generated a knock-in mouse with rhodopsin replaced by its E122Q mutant, which exhibits properties different from those of wild-type (WT) rhodopsin. Knock-in mouse rods with E122Q rhodopsin exhibited a photosensitivity about 70% of WT. Correspondingly, their single-photon response had an amplitude about 80% of WT, and a rate of decline from peak about 1.3 times of WT. The overall 30% lower photosensitivity of mutant rods can be explained by a lower pigment photosensitivity (0.9) and the smaller single-photon response (0.8). The slower decline of the response, however, did not correlate with the 10-fold shorter lifetime of the meta-II state of E122Q rhodopsin. This shorter lifetime became evident in the recovery phase of rod cells only when arrestin was absent. Simulation analysis of the photoresponse profile indicated that the slower decline and the smaller amplitude of the single-photon response can both be explained by the shift in the meta-I/meta-II equilibrium of E122Q rhodopsin toward meta-I. The difference in meta-III lifetime between WT and E122Q mutant became obvious in the recovery phase of the dark current after moderate photobleaching of rod cells. Thus, the present study clearly reveals how the molecular properties of rhodopsin affect the amplitude, shape, and kinetics of the rod response.     

Expression profiles of Insulin-like growth factor binding protein-like 1 in the developing mouse forebrain

Insulin-like growth factor binding protein-like 1 (IGFBPL-1) has a domain structure that resembles one in the insulin-like growth factor binding proteins (IGFBPs), which is a domain known to be bound by insulin-like growth factors (IGFs) with high affinity and to thereby affect cell growth and metabolism. However, the distribution and functions of IGFBPL-1 in the brain are not known. Thus, to clarify IGFBPL-1 expression profiles during development, we have investigated the expression patterns of IGFBPL-1 mRNA in developing mouse brains using in situ hybridization, focusing especially on the forebrain. On embryonic day (E)12, IGFBPL-1mRNA expressions were localized in the preplate of the neopallium, dorsal thalamus, the differentiating field of the epithalamus, and the hippocampus. On E14, strong expression was recognized in the subventricular zone (SVZ) and intermediate zone (IMZ) in the neopallium, hippocampus, the differentiating field of the epithalamus, and the dorsal thalamus. E14 expressions were generally the strongest among all the stages examined and signals decreased thereafter. At E16 and E18, IGFBPL-1mRNA was strongly detected in the hippocampus and the SVZ of the neopallium. In postnatal stages, mRNA was found only in the granule cell layer of the dentate gyrus (DG). Notably, at P20, expression was restricted to the deep fraction of the granule cell layer, the location of late-born DG neurons.