Purification, characterization and gene expression of a glycine and proline-rich antibacterial protein family from larvae of a beetle, Allomyrina dichotoma.

Two structurally related antibacterial proteins were isolated from larvae of a beetle, Allomyrina dichotoma, immunized with Escherichia coli. The two proteins were designated A. dichotoma (A. d.) coleoptericin A and B. The mature portion of A. d. coleoptericins deduced from nucleotide sequences of the cDNAs consists of seventy-two amino acids without cysteine residues and is rich in glycine (11.1%) and proline (11.1%). Comparison of the amino acid sequences of the A. d. coleoptericins revealed that these antibacterial proteins have 94%, 75%, 50% and 43% similarity to rhinocerosin, holotricin 2, coleoptericin and acaloleptin A1. Recombinant A. d. coleoptericin A and B showed strong antibacterial activity against Staphylococcus aureus, methicillin resistant S. aureus (MRSA) and Bacillus subtilis. Recombinant A. d. coleoptericin A and B were shown to not form pores through bacterial membranes of E. coli, but to hamper cell division. Results of Northern blotting showed that A. d. coleoptericin genes are inducible by bacteria and are expressed strongly in the fat bodies and haemocytes, and weakly in the Malpighian tubules. Analysis of the evolutionary relationship of amino acid sequences among A. d. coleoptericins and other antibacterial proteins suggests that A. d. coleoptericins, rhinocerosin and holotricin 2 are closely related and form a gene family.

Characterization of the gammac chain among 27 unrelated Japanese patients with X-linked severe combined immunodeficiency (X-SCID).

X-linked severe combined immunodeficiency (X-SCID) is a rare fatal disease that is caused by mutations in the gene encoding the gammac chain. In this study, 27 unrelated Japanese patients with X-SCID were examined in terms of their genetic mutations and surface expression of the gammac chain. Among 25 patients examined, excluding two patients with large deletions, 23 different mutations were identified in the IL2RG gene, including 10 novel mutations. One patient bearing an extracellular mutation and all three of the patients bearing intracellular mutations after exon 7 expressed the gammac chain on the cell surface. Overall, 84% of patients lacked surface expression of the gammac chain leading to a diagnosis of X-SCID.

Cerebral vasculitis in chronic mucocutaneous candidiasis: autopsy case report.

An autopsy case of chronic mucocutaneous candidiasis (CMCC) is reported here, in which cerebral vasculitis developed in the final stage. A 32-year-old man who had suffered from superficial candidial infection since his childhood was diagnosed as having CMCC. During the past 7 years the patient had developed various associated disorders including insulin-dependent diabetes mellitus (IDDM), common variable immunodeficiency (CVID), candidial esophagitis, multiple digestive tract ulcers and pyothorax. In 1998, at the age of 32, he developed convulsions that were accompanied by impairment of consciousness, and which were temporarily treated with steroid pulsed-medication. Epileptic status associated with widespread cerebral infarctions occurred subsequently, however, and the patient died of sepsis 2 months later. At autopsy, multiple cerebral infarctions and arterial thrombosis were evident. These were histologically proven to be primary vasculitis which was confined solely to the brain, and this was verified by general pathological examination. Thus, some as yet unknown cerebrovascular factors might be involved in the onset of an autoimmune-related vasculitis in patients with a longstanding immunodeficiency state such as CMCC.

Isolation and characterization of a new member of the insect defensin family from a beetle, Allomyrina dichotoma.

A new family member of insect defensin, an antibacterial peptide, has been isolated from larvae of a beetle, Allomyrina dichotoma. The peptide consisted of 43 amino acids and 6 cystein residues were conserved in the same position as that of other insect defensins. The new defensin was found to be inducible by bacterial injection. Analysis of the antibacterial spectrum of A. dichotoma defensin indicated that this peptide showed antibacterial activity against Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis but not against Gram-negative bacteria like Escherichia coli and Pseudomonas aeruginosa, indicating a typical spectrum of the insect defensin family. In addition, A. dichotoma defensin also exhibited antibacterial activity against methicillin-resistant S. aureus (MRSA) isolated from a patient.

Nucleotide sequence of 5'-upstream region and expression of a silkworm gene encoding a new member of the attacin family.

A genomic clone encoding a new member of attacin, an insect antibacterial protein, was isolated from a genomic library of the silkworm, Bombyx mori, and the nucleotide sequence of the 5'-upstream region was determined. The region contained Bm 1, a highly repetitive element of B. mori and a lipopolysaccharide (LPS) response element (RE)(NF-kappaB binding site), CAAT box and TATA box. Northern blot analysis showed that the attacin gene expression was rapidly induced by bacterial cell wall components such as LPS from Escherichia coli and peptidoglycan (PG) from Micrococcus luteus, suggesting that attacin plays an important role in an early phase of the self-defense system upon bacterial infection.

Effect of adenosine on phorbol myristate acetate induced-reactive oxygen metabolite production in cultured mesangial cells.

Cultured rat mesangial cells produced chemiluminescence in response to phorbol myristate acetate. Because 90% of this chemiluminescence was suppressed by 10 mU/ml superoxide dismutase, reactive oxygen metabolites are most likely involved in the chemiluminescence reaction. To clarify the role of cyclic AMP in the regulation of reactive oxygen metabolites production in cultured mesangial cells, we studied the effects of adenosine, an adenosine analog (2-chloroadenosine), and forskolin on phorbol myristate acetate-induced chemiluminescence. Exogenous adenosine suppressed the production of reactive oxygen metabolites in a dose-dependent manner (maximum at 1 mM adenosine: 70.6 +/- 3.2% of control). Lower concentration of 2-chloroadenosine (maximum at 100 mu M 2-chloroadenosine: 63.2 +/- 2.1% of control) and forskolin (maximum at 200 mu M forskolin: 53.4 +/- 2.6% of control) also significantly suppressed the production of reactive oxygen metabolites. In addition, adenosine analogs increased intracellular cyclic AMP in a dose-dependent manner in the order: 5'-N-ethylcarboxamidoadenosine>2-chloroadenosine>N6-cyclohexyladenosine. These results are in accordance with the probability that exogenous adenosine, by increasing intracellular cyclic AMP via the A2 receptor, inhibits the production of reactive oxygen metabolites through direct protein kinase C activation induced by phorbol myristate acetate in cultured rat mesangial cells.

Microcephaly and early-onset nephrotic syndrome--confusion in Galloway-Mowat syndrome.

We report a 2-year-old girl with nephrotic syndrome, microcephaly, seizures and psychomotor retardation. Histological studies of a renal biopsy revealed focal glomerular sclerosis with mesangiolysis and capillary microaneurysms. Dysmorphic features were remarkable: abnormal-shaped skull, coarse hair, narrow forehead, large low-set ears, almond-shaped eyes, low nasal bridge, pinched nose, thin lips and micrognathia. Cases with this rare combination of microcephaly and early onset of nephrotic syndrome with various neurological abnormalities have been reported. However, clinical manifestations and histological findings showed a wide variation, and there is a lot of confusion in this syndrome. We therefore reviewed the previous reports and propose a new classification of this syndrome.

Occurrence of novel types of nitric oxide synthase in the silkworm, Bombyx mori.

Nitric oxide (NO) synthase activity was detected in fat body and the Malpighian tubles of the silkworm, Bombyx mori. Main NO synthase activity in the fat body was Ca(2+)/calmodulin-dependent, inducible by bacterial lipopolysaccharide (LPS) and required NADPH, FAD, FMN, dithiothreitol (DTT) and tetrahydrobiopterin (BH4) as cofactors for the full expression of the activity. The Malpighian tubles contained two types of NO synthase. One was Ca(2+)-independent, calmodulin-dependent and constitutive and the other was Ca(2+)-dependent and constitutive. The former NO synthase required the same cofactors as fat body NO synthase. The activity of Malpighian tuble NO synthases increased dramatically at the end of the last instar period, just prior to spinning. These results indicate that B. mori contains new types of NO synthase, suggesting the wide distribution and different characteristics of this enzyme among vertebrates and invertebrates.

Molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney.

Electrically silent Na(+)-(K+)-Cl- transporter systems are present in a wide variety of cells and serve diverse physiological functions. In chloride secretory and absorbing epithelia, these cotransporters provide the chloride entry mechanism crucial for transcellular chloride transport. We have isolated cDNAs encoding the two major electroneutral sodium-chloride transporters present in the mammalian kidney, the bumetanide-sensitive Na(+)-K(+)-Cl- symporter and thiazide-sensitive Na(+)-Cl- cotransporter, and have characterized their functional activity in Xenopus laevis oocytes. Despite their differing sensitivities to bumetanide and thiazides and their different requirements for potassium, these approximately 115-kDa proteins share significant sequence similarity (approximately 60%) and exhibit a topology featuring 12 potential membrane-spanning helices flanked by long non-hydrophobic domains at the NH2 and COOH termini. Northern blot analysis and in situ hybridization indicate that these transporters are expressed predominantly in kidney with an intrarenal distribution consistent with their recognized functional localization. These proteins establish a new family of Na(+)-(K+)-Cl- cotransporters.

Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter.

Electroneutral Na+:Cl- cotransport systems are involved in a number of important physiological processes including salt absorption and secretion by epithelia and cell volume regulation. One group of Na+:Cl- cotransporters is specifically inhibited by the benzothiadiazine (thiazide) class of diuretic agents and can be distinguished from Na+:K+:2Cl- cotransporters based on a lack of K+ requirement and insensitivity to sulfamoylbenzoic acid diruetics like bumetanide. We report here the isolation of a cDNA encoding a thiazide-sensitive, electroneutral sodium-chloride cotransporter from the winter flounder urinary bladder using an expression cloning strategy. The pharmacological and kinetic characteristics of the cloned cotransporter are consistent with the properties of native thiazide-sensitive sodium-chloride cotransporters in teleost urinary bladder and mammalian renal distal tubule epithelia. The nucleotide sequence predicts a protein of 1023 amino acids (112 kDa) with 12 putative membrane-spanning regions, which is not related to other previously cloned sodium or chloride transporters. Northern hybridization shows two different gene products: a 3.7-kb mRNA localized only to the urinary bladder and a 3.0-kb mRNA present in several non-bladder/kidney tissues.

Intranephron PGE2 production in stroke-prone spontaneously hypertensive rats.

To investigate whether intranephron prostaglandin E2 (PGE2) production in stroke-prone spontaneously hypertensive rats (SHRSP) differs from that in Wistar-Kyoto rats (WKY), we measured PGE2 accumulation rates in microdissected nephron segments from 4- to 6- and 12- to 14-wk-old male rats by radioimmunoassay. In both young and adult WKY, PGE2 accumulation was highest in the papillary collecting duct (PCD) and outer medullary and cortical collecting tubules, intermediate in the glomerulus (Glm), medullary and cortical thick ascending limbs of Henle's loop, and distal tubule, and negligible in the proximal tubule. PGE2 accumulation in adult WKY was severalfold higher than that in young WKY. PGE2 accumulation in adult and prehypertensive young SHRSP was significantly lower than that of respective WKY in most segments, whereas urinary PGE2 excretion was significantly higher in SHRSP than in age-matched WKY. Plasma arginine vasopressin concentrations in adult SHRSP were significantly higher than in WKY. PGE2 accumulation stimulated by 5 microM arachidonic acid was significantly lower in SHRSP than in WKY in most segments of young rats but was lower only in Glm and PCD of adult rats. PGE2 accumulation stimulated by 2 microM Ca2+ ionophore A23187 was significantly lower in most segments of adult and young SHRSP. These results indicate that a decrease in renal tubular PGE2 productive activities in SHRSP might not be caused by secondary adaptation to hypertension.

Furosemide directly stimulates prostaglandin E2 production in the thick ascending limb of Henle's loop.

Studies were conducted to investigate direct effects of loop diuretics on prostaglandin E2 (PGE2) production using microdissected nephron segments. At first, the effect of indomethacin on the diuretic response to furosemide was re-evaluated in anesthetized rats. Indomethacin significantly attenuated the diuretic, natriuretic and chloruretic effects of furosemide without significantly affecting inulin and p-aminohippurate clearance or filtration fraction. But, in nondiuretic states, indomethacin had no significant effects on these parameters. Furosemide, ethacrynic acid and bumetanide significantly increased PGE2 production in cortical and medullary thick ascending limbs of Henle's loop (P less than .001), but not PGE2 production in the cortical and outer medullary collecting tubules. The effect of furosemide on PGE2 production in CTAL was dose-dependent, and higher concentrations of of furosemide than 10(-6) M significantly increased PGE2 production. On the other hand, chlorothiazide showed no PGE2 productive stimulation in these four nephron segments. This study demonstrates that the enhanced PGE2 production in the thick ascending limb of Henle's loop by furosemide and other loop diuretics is one possible mechanism of these drugs.

Inhibitory effect of cyclic AMP on phorbol ester-stimulated production of reactive oxygen metabolites in rat glomeruli.

Studies were conducted to investigate cross-talk between protein kinase C (PKC) and cyclic AMP (cAMP) pathways using rat glomeruli (Glm). Phorbol 12-myristate 13-acetate (PMA), a PKC activator, stimulated production of reactive oxygen metabolites (ROM) in Glm. Forskolin and dibutyryl cAMP (Bt2cAMP) inhibited production of ROM dose-dependently. In the presence of both Bt2cAMP and 3-isobutyl-1-methylxanthine (IBMX) an additive effect was observed. Forskolin at 10(-4) inhibited translocation of PKC from the cytosol to the membrane. These results demonstrate that cAMP-mediated inhibition can occur at a step distal to PKC activation.

[Clinical studies of SM-4300 in children].

In vitro experiment with phagocytic and bactericidal activity of human neutrophils, SM-4300 augmented opsonic activity against Pseudomonas aeruginosa. After a single dose of 200 mg/kg body weight SM-4300 to a child with common variable immunodeficiency (CVI), serum IgG concentration/time curve was similar to that after PEG-Ig administration. In combination therapies with SM-4300 and antibiotics in 8 children with bacterial infections, beneficial efficacy of SM-4300 was observed. There was no adverse reaction in 16 times injections to 9 children with bacterial infections and 5 times to a child with CVI.