Dynamic alteration of human β-defensin 2 localization from cytoplasm to intercellular space in psoriatic skin

Defensins are cationic antimicrobial peptides with a broad spectrum. Recently human beta-defensin 2 (hBD-2) has been isolated from psoriatic skin; however, its exact localization and fate have not been fully understood. We studied the distribution pattern of hBD-2 in skin tissues of psoriasis and other inflammatory skin diseases. In the upper spinous and granular layer of psoriasis vulgaris hBD-2 was present in the cytoplasm. In the horny layer the positive signals were in a basket-weave pattern, indicating possible accumulation of hBD-2 in the intercellular space. The similar pattern of hBD-2 distribution was observed in the lesions of nummular eczema and atopic dermatitis. hBD-2 was not detected in the section of normal elbow and knee skin. When isolated psoriatic scales were stained, hBD-2 was detected in a wrapping paper-like distribution pattern surrounding the corneocytes. In horny layer of psoriatic skin hBD-2 was closely associated or colocalized with elafin, which is known to be in extracellular space, as demonstrated by double staining. Western blot analysis using cultured human keratinocytes detected hBD-2 with an expected size in the conditioned medium and in the cell lysates when stimulated with 5% FCS or IL-alpha. These results indicate that hBD-2 was synthesized and remained in cytoplasm in the upper spinous and granular layer, and then secreted into intercellular space in the horny layer. This dynamic change in hBD-2 distribution in epidermis is certainly relevant to function as an innate host defense mechanism against invading micro-organisms.     

Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia

Clostridium botulinum serotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.     

Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse development

The cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 are thought to regulate progression of the cell cycle. We have previously shown that the phenotypes of p27-/- mice are substantially different from those of p57-/- mice, suggesting that spatial and temporal expression patterns of p27Kip1 and p57Kip2 might be distinct. In this study, the roles of p27Kip1 and p57Kip2 in development were examined by characterizing their expression patterns during mouse embryogenesis by immunohistochemical analysis. Whereas certain organs and tissues (brain, lens, ganglion, lung, heart, liver, skin and kidney) expressed both proteins, others expressed only p27Kip1 (thymus, spleen, retina, testis and ovary) or only p57Kip2 (gut, palate, pancreas, cartilage and skeletal muscle). In addition, some organs expressed both p27Kip1 and p57Kip2 but showed mutually exclusive patterns of distribution among tissues. Thus, in the adrenal gland, p57Kip2 was expressed in the cortex but not in the medulla, whereas p27Kip1 was expressed in the medulla but not in the cortex. Whereas the expression of p57Kip2 in most tissues was restricted to embryogenesis, expression of p27Kip1 in many tissues was maintained in adult animals. Double-label immunofluorescence staining with either anti-p27Kip1 or anti-p57Kip2 and anti-BrdU revealed that the expression of p27Kip1 and p57Kip2 was inversely correlated with cell proliferation, suggesting that p27Kip1 and p57Kip2 are expressed exclusively in postmitotic cells. These complex spatial and temporal patterns of expression are consistent with the phenotypes of mice deficient in p27Kip1 or p57Kip2, and they suggest that these proteins might play important roles in tissue development.     

Effects of long-term acidification of extracellular pH on ATP-induced calcium mobilization in rabbit lens epithelial cells

ATP-induced calcium (Ca2+) mobilization was investigated in rabbit lens epithelial cells that had been cultured in a medium with pH of 7.4 (group 1), 7.2 (group 2), or 7.0 (group 3) for 10 to 21 d. Intracellular free Ca2+ ([Ca2+]i and pH (pHi) were measured by using fluorescent dyes, fura-2 and BCECF, respectively. The long-term acidification decreased the pHi to 7.15 +/- 0.01, from 7.22 +/- 0.01, in group 2 and to 7.09 +/- 0.01 in group 3. The administration of 10 micromol/l ATP produced an initial peak followed by a sustained increase in [Ca2+]i in the lens cells of group 1. Both the initial peak and the sustained increase in [Ca2+]i were enhanced in groups 2 and 3. The initial peak was abolished by pretreatment with 1 micromol/l thapsigargin, an ER Ca2+ pump inhibitor, but was not affected by the removal of extracellular Ca2+. On the other hand, the sustained increase was suppressed either by the thapsigargin treatment or by the Ca2+ removal. Treatment with only thapsigargin caused a sustained increase in [Ca2+]i that was greater in group 3 than in group 1. These results suggest that (1) the ATP-induced initial peak in [Ca2+]i is due to Ca2+ release from the intracellular stores, (2) the sustained increase in [Ca2+]i is mediated through either Ca2+ influx from the extracellular space or Ca2+ release from the store triggered by the Ca2+ influx, and (3) long-term, moderate acidification enhances both the initial peak and the sustained increase in [Ca2+)]i in rabbit lens epithelial cells. One possible mechanism of the ATP-induced Ca2+ influx seems to be a capacitative Ca2+ entry pathway.     

A case of the esophageal candidiasis supposedly caused by rhinenchysis steroid chronic administration before sleep]

In general, steroid is mainly used as anti-inflammatory action in case of allergic diseases. As one of the side effects of inhalation steroid, a report is given below regarding buccal capsule/esophageal candidiasis. The patient came to the hospital with the chief complaint regarding passage dysphagia in the time of deglutition; pharyngitis and esophageal candidiasis were found by endoscopy of upper gastrointestinal tract.The interview after the endoscopy revealed that the patient, a 69-year-old female was diagnosed as chronic perennial allergic rhinitis a few years ago, and had been inhaling rhinenchysis Beclometasone dipropionate (BDP) before sleep every day for the past two years because using this collunarium seemed to mitigate the nasal obstruction and mucus during sleep. The patient did not report this fact before the endocsopy because she did not associate it with her subjective symptom. In this case, it was assumed that nebulized rhinenchysis BDP was accidentally swallowed to the pharynx and esophagus during sleep. As a treatment, rhinenchysis BDP was canceled and instead Azunol mouth washing (gargling/nasal douche) was used. No antifungal agent was used. In two weeks, the patient reported some improvement, and this was confirmed by reexamination of the upper gastrointestinal tract using endoscope in one month and a half. Pharyngitis was improved, and in the digital endoscopic assessment of esophageal candidiasis complicating inhaled steroid therapy the esophageal candidiasis became Grade I (mild grade). As for the later progress, the patient did not report any subjective symptoms such as nasal obstruction and dysphagia. In addition, the inflammation caused by candidiasis and found in the early examination was improved. The patient in this case was under treatment for thrombosis in the vein of lower extremity, but no complications such as diabetes mellitus or immune deficiency syndrome were observed. DISCUSSION: Esophageal candidiasis by chronic administration of inhalation of steroid before sleep for asthmatic patients has been reported. However, there has not been a report of esophageal candidiasis by chronic administration of rhinenchysis steroid before sleep for patients with allergic rhinitis. Similarly, in the case of the use of steroid in the form of collunarium before sleep, steroid stayed in the esophagus via the transendothelial nasal cavity, and that seemed to cause, in the long run, to develop esophageal candidiasis. CONCLUSIONS: One of the implications of the above case is that collunarium can go down, even when it is nebulized in the nasal cavity, to the esophagus via the nasal cavity to buccal capsule. This suggests the necessity for preventative measures in the case of chronic administration of steroid as follows. A. Blowing of the nose just after the use of collunarium B. Daily rinsing (gargling and nasal douche).     

Ex vivo localization of the mouse saposin C activation region for acid beta-glucosidase

Saposin C is a biological activator of acid beta-glucosidase (GCase), the lysosomal hydrolase with activity towards glucosylceramide (GC). In addition, saposin C possesses a functional domain that determines the in vitro and ex vivo neuritogenic effects of prosaposin, the precursor of saposins A, B, C, and D. The domains for enzymatic activation and neuritogenic function segregate in vitro, respectively, to the carboxyl- and amino-terminal halves of human and mouse saposin C. A chimeric mouse saposin C(1-8)B(8-28)C(30-80) was created to obliterate the neuritogenic region by substituting amino acids 9-29 of saposin C with amino acids 8-28 of saposin B. This saposin showed normal in vitro enzymatic activation effects toward GCase, but no neuritogenic activity. An altered prosaposin was made to contain the chimeric saposin C region. Expression of this altered or wild-type prosaposin was driven by the PGK-1 promoter as a transgene in prosaposin knock-out mice. In cultured fibroblasts from such mice, expressed saposins localized to the lysosomal compartments. Metabolic lipid labeling using L-[3-(14)C]serine showed retention or clearance of GC in prosaposin deficient or transgene reconstituted cells, respectively. In addition, sulfatide catabolism, that requires saposin B and arylsulfatase, was also normalized in prosaposin KO cells reconstituted with the transgenes. These data show that the transgenic prosaposins were expressed and processed to functional saposins in fibroblasts. These results also show that the enzymatic activation domain is located at carboxyl-terminal half of saposin C and functions only in the context of the general saposin structure.     

Neural fibrolipoma of the superficial peroneal nerve in the ankle: a case report with immunohistochemical analysis

This report presents a case of neural fibrolipoma arising from the superficial peroneal nerve in the ankle. A 28-year-old woman was referred with a soft tissue mass in the anterior aspect of the right ankle, which had been gradually enlarging for the past 10 years. Magnetic resonance imaging showed a mass lesion, measuring approximately 8 x 3 x 2 cm, with high to partially low signal intensity on both T1- and T2-weighted images. A band of low signal intensity within the lesion, which is indicative of coexistence with the tumor and the superficial peroneal nerve, could be detected on both T1- and T2-weighted images. The patient underwent an excisional biopsy. The specimen microscopically consisted of nerve bundles and fibro-fatty proliferation with abundant collagen fibers. Immunoreactivity for CD34 antigen antibody was detected in fibrous spindle cells. This is the first report to present an immunohistochemical profile of neural fibrolipoma. Neural fibrolipoma should be considered as a differential diagnosis when a lipomatous lesion is encountered in the foot or ankle as well as in the upper extremities.     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA

The livers of DNase II-deficient mouse embryos contain many macrophages carrying undigested DNA, and the embryos die in utero. Here we report that erythroid precursor cells underwent apoptosis in the livers of DNase II-deficient embryos and that in the liver, interferon-beta mRNA was expressed by the resident macrophages. When the DNase II-deficient mice were crossed with mice deficient in type I interferon receptor, the resultant 'double-mutant' mice were born healthy. The double-mutant embryos expressed interferon-beta mRNA, but the expression of a subset of the interferon-responsive genes dysregulated in DNase II-deficient embryos was restored to normal. These results indicate that the inability to degrade DNA derived from erythroid precursors results in interferon-beta production that induces expression of a specific set of interferon-responsive genes associated with embryonic lethality in DNase II-deficient mice.