cDNAs encoding chitinases were cloned and characterized from Bombyx mori and Hyphantria cunea, and their gene expression during the metamorphosis was also studied. The chitinase cDNA from B. mori encodes a protein of 565 amino acids with a calculated molecular mass of 63.4 kDa and the H. cunea chitinase cDNA encodes a protein of 553 amino acids with a calculated molecular mass of 62.0 kDa. Amino acid alignment of the two chitinases revealed 75% homology and 77-80% with M. sexta chitinase. The putative cleavage site of the signal peptide was between amino acid residues 20 and 21 for both chitinases. There were three potential N-glycosylation sites in the chitinase of B. mori at the amino acid residues 86-89, NFTS 304-307, NATG, 398-401, NYTV, whereas two potential N-glycosylation sites were present at the amino acid residues 86-89, NFTA and 304-307, NATG, in that of H. cunea. Southern blot analysis of total genomic DNA suggested that the B. mori genome has only one chitinase gene detectable by the cDNA probe and the H. cunea genome has one or two chitinase gene copies. Northern analysis indicated that gene expression was up-regulated during the molting process, larval-pupal transformation and pupal-adult transformation, when enzymatic degradation of cuticle was occurring.

Amino Acid Sequence↗

PubMed1998

Isolation and characterization of immune-related genes from the fall webworm, Hyphantria cunea, using PCR-based differential display and subtractive cloning.

Following injection of bacteria into the hemocoel of the fall webworm, Hyphantria cunea, several inducible genes were identified and characterized using PCR-based differential display (DD-PCR) and subtractive cloning. Ten immune-related cDNA clones (Hdd1, Hdd2, Hdd3, Hdd11, Hdd13, Hdd15, Hdd17, Hdd23, Hs106, Hs302) were isolated and characterized. The deduced amino acid sequence of Hdd2 was shown to be a member of the copper, zinc superoxide dismutase (Cu-Zn SOD) family. The H. cunea Cu-Zn SOD is novel in that it is up-regulated following a bacterial challenge and has a putative signal peptide suggesting its secretion and involvement in the insect immune response. Hdd3 was found to encode a new member of the serpin (serine protease inhibitor) family. The putative lectin corresponding to Hdd15 is of a different kind in that it has two lectin C domains in a single molecule. These two lectin C domains show significant homology to the lectin C domain of Periplaneta lipopolysaccharide binding protein (LPS-BP). Three cloned genes, Hdd17, Hs106 and Hs302, encode a homologue to Bombyx mori Gram negative binding protein, a hemolin-like protein and a attacin-like protein, respectively. The deduced amino acid sequences from Hdd11 showed weak homology with a Locusta migratoria hemolymph protein. On the contrary, Hdd1, Hdd13 and Hdd23 did not reveal any significant homology with known proteins. All of the 10 genes were clearly inducible by E. coli and M. luteus injection. Injection of distilled water only slightly induced mRNA levels. Comparison of temporal mRNA expression following E. coli injection showed three types of expression patterns.

Amino Acid Sequence↗

PubMed1998

Cre/loxP-mediated excision and amplification of large segments of the Escherichia coli genome.

The isolation and amplification of large, predetermined segments of a genome from its host have been explored. The prototype of our approach was the excisional replication of some viruses such as the lambda-lysogen. Similar machinery was used to excise and amplify large genomic segments of Escherichia coli in its host. Two loxP sequences for a site-specific recombinase Cre, together with a conditional replication origin (pi-dependent gamma-ori), were inserted into the genome by homologous recombination at predetermined sites, 50-100 kb apart. Cre and pir200 which encodes the site-specific recombinase Cre and an ori-specific replication protein pi, respectively, were also introduced into the genome. The predetermined genomic segments flanked by the loxP sequences were excised and amplified upon induction of the cre and pir200 genes which were under the control of the tet promoter. This excised and amplified DNA could be easily purified as a large plasmid. This procedure can provide an alternative to conventional cloning methods by obtaining predetermined large genomic segments directly from the original organisms. In this study, using the Cre/loxP site-specific recombination and pi/gamma-ori replication system of plasmid R6K, a procedure was devised that could isolate a large segment of the E. coli genome and demonstrated the feasibility of the procedure by excising and amplifying the 50-kb trg-narZ and 100-kb trg-hipA regions of the E. coli W3110 genome.

Bacterial Proteins↗

PubMed1998

Localized mucosal involvement and severe pulmonary involvement in a young patient with paraneoplastic pemphigus associated with Castleman's tumour.

We describe a 19-year-old female patient who developed recurrent ulcerations limited to the orogenital mucosa for the last 3 years. She also developed dyspnoea 5 months after the onset of the orogenital lesions. Castleman's tumour of the retroperitoneum was found incidentally during routine physical examination. The diagnosis of paraneoplastic pemphigus (PNP) was made by pathological and immunological studies. The orogenital ulceration responded well to corticosteroid therapy, but severe bronchiolitis obliterans progressed despite intensive care. The patient eventually died from respiratory failure. This case demonstrates the diversity of clinical features of paraneoplastic pemphigus.

Adult↗

PubMed1998

Effects of nitrogenous components of urine on sperm motility: an in vitro study.

The effects of the major nitrogenous components of urine (ammonia, creatinine, urea and uric acid) on sperm motility were investigated. Semen samples were obtained by masturbation from a young volunteer with a normal spermiogram and fertility. Sperm motility was measured using a Sperm Quality Analyzer and Makler Chamber. When semen was mixed with a urine sample for which acidity and osmolality were adjusted to pH 7.5 and 340 mOsm/kg, the higher the concentration of the urine, the poorer the sperm motility. When solutions of various concentrations of the nitrogenous compounds which are excreted in urine were mixed with semen, sperm motility decreased in proportion to ammonia levels and was markedly decreased after 30 min. However, in the case of creatinine, urea and uric acid, good motility was maintained for 30 min regardless of their amounts. In conclusion, urine ammonia content is detrimental to sperm motility and may be an important factor in retrograde ejaculation.

Adult↗

PubMed1998

Epidermolysis bullosa acquisita localized to the face.

A 39-year-old woman had a three-year history of recurrent bullous eruption localized to her left cheek. The diagnosis of epidermolysis bullosa acquisita was confirmed by means of direct immunofluorescence and direct immunoelectron microscopic studies performed on the perilesional salt-split skin. Topical corticosteroid treatment reduced pruritus and bullae formation. This case of localized epidermolysis bullosa acquisita on the face is reminiscent of Brunsting-Perry cicatricial pemphigoid. We also review the previously reported cases of localized epidermolysis bullosa acquisita.

Administration, Topical↗

PubMed1998

Multiple lentigines syndrome: a comparison of normal skin and lentiginous skin by electron microscopy and immunohistochemical staining.

A 26-year-old Korean man exhibited generalized lentigines, a slight prominence of the chin, short stature, and a familial history consistent with the autosomal dominant mode of inheritance. The multiple lentigines appeared at the age of 1 and spread over his entire body including the scalp, palms, and soles by the age of 4. There were several large dark pigmented spots on the trunk, but no other skin abnormalities were found. We performed histologic studies of the normal skin and the lentigo by both electron and light microscopy with immunohistochemical stains. Routine hematoxylin-eosin staining of the pigmented skin revealed heavily pigmented basal and spinous layers and prominent rete ridges. Fontana-Masson stain showed enormous black pigmentations on the basal and spinous layers of the pigmented macule. S-100 protein stain of the pigmented lesion demonstrated an increased number of melanocytes per unit skin area. Ultrastructural studies showed the presence of extensive aggregated melanosomes within the keratinocytes in the epidermis of lentigo, but not the presence of giant melanosomes in either normal skin or lentigines.

Adult↗

PubMed1998

Effect of stem cell factor, interleukin-6, nitric oxide and transforming growth factor-beta on the osteoclast differentiation induced by 1 alpha,25-(OH)2D3 in primary murine bone marrow cultures.

Osteotropic hormones and cytokines are involved in the differentiation of osteoclast progenitors from haematopoietic stem cells to multinucleated osteoclasts which mediate bone resorption. Stem cell factor, interleukin-6, nitric oxide, and transforming growth factor-beta are implicated in the regulation of bone resorption by osteoclast. We test whether stem cell factor, interleukin-6, nitric oxide, and transforming growth factor-beta affect the generation of osteoclast-like multi-nucleated cells induced by 1 alpha,25-(OH)2D3. 1 alpha,25-(OH)2D3 increase the generation of osteoclast-like cells retaining osteoclast characteristics including multinuclearity and positive staining for tartrate-resistant acid phosphatase. Combined treatment of stem cell factor with interleukin-6 synergistically potentiates the ability of 1 alpha,25-(OH)2D3 to generate tartrate-resistant acid phosphatase-positive multinucleated cells. However, either stem cell factor or interleukin-6 alone does not induce the generation of tartrate-resistant acid phosphatase-positive multinucleated cells. Transforming growth factor-beta produces a biphasic effect on osteoclast generation induced by 1 alpha,25-(OH)2D3. Transforming growth factor-beta stimulates osteoclast generation at low concentration (0.1 ng/ml) whereas it suppresses the formation of osteoclast-like cell at higher concentration (1 ng/ml). Sodium nitroprusside, a donor of nitric oxide, almost completely inhibits the generation of 1 alpha,25-(OH)2D3-induced osteoclast at high concentration (100 microM), but it significantly enhances the osteoclast generation at low concentrations (3 microM). These results suggest that stem cell factor, interleukin-6, transforming growth factor-beta, and nitric oxide interact with 1 alpha,25-(OH)2D3 to modulate the differentiation of hematopoietic precursors toward committed osteoclast precursors.

24,25-Dihydroxyvitamin D 3↗