Potential roles of histones in host defense as antimicrobial agents.

Antimicrobial peptides (AMPs), which are widely distributed in various organisms, comprise part of the host innate defense system to kill or damage bacterial and fungal pathogens. Amphibian skin is known to produce various AMPs, and is used as a source material in attempts to identify novel therapeutic AMPs. More than one hundred frog AMPs have been identified to date. In our previous study, we isolated histone H2B with antibacterial properties from the skin of the Schlegel's green tree frog Rhacophorus schlegelii. Although antimicrobial histone H2B has not been obtained from the skin of any species other than R. schlegelii, histones and histone-derived fragments with antimicrobial activities have been found in some specific cells of a diverse range of organisms from shrimps to humans. At least a portion of these fragments are known to be produced from "precursor histones" via specific cleavage by endogenous proteases. These antimicrobial histones and the fragments that act as physiological barriers of cells have a variety of antimicrobial actions and functions, including bacterial cell membrane permeabilization, penetration into the membrane followed by binding to bacterial DNA and/or RNA, binding to bacterial lipopolysaccharide (LPS) in the membrane, neutralizing the toxicity of bacterial LPS, and entrapping pathogens as a component of neutrophil extracellular traps (NETs). This review discusses the literature regarding the isolation, antimicrobial properties, and modes of action of antimicrobial histones and fragmented histones along with a brief introduction of typical amphibian skin AMPs.

Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris.

Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.

Incidence of bone fracture in patients receiving luteinizing hormone-releasing hormone agonists for prostate cancer.

OBJECTIVE: To investigate the incidence of bone fractures in patients receiving luteinizing hormone-releasing hormone agonists (LHRH-a) for prostate cancer (in whom a continued low testosterone level after the long-term administration of these drugs reduces bone mineral density), and thus determine the risk of secondary osteoporosis. PATIENTS AND METHODS: Between 1994 and 1999, 218 patients (mean age 77.3 years) were treated for >/= 6 months with LHRH-a for prostate cancer; of these, 14 (6%) had a bone fracture during their treatment. Patients with fracture associated with motor vehicle accidents were excluded. The bone density in the third lumbar vertebra was meas-ured using quantitative computed tomography. Osteocalcin, 1,25-(OH)2 vitamin D, urinary type 1 collagen cross-linked N-telopeptides (NTx), parathyroid hormone and calcitonin were measured as metabolic markers. RESULTS: The mean age of the patients with fracture was 78 years; the mean (range) interval from the start of treatment to fracture was 28 (11-46) months. There was no case of a bone fracture at the site of a metastasis from prostate cancer. The bone density was significantly lower in the patients with a fracture than in those without. Of the bone metabolic markers, NTx was higher in those with a fracture. CONCLUSION: There is a need to measure bone mineral density and bone metabolic markers periodically, and to evaluate secondary osteoporosis in patients receiving long-term LHRH-a for prostate cancer.

[A clinical study of secondary osteoporosis induced by endocrine therapy for prostate cancer].

PURPOSE: There is one of the big problems that endocrine therapy for prostate cancer causes to induce secondary osteoporosis. The risk factors and future treatments for osteoporosis were investigated. MATERIALS AND METHODS: 31 patients treated with luteinizing hormone releasing hormone agonists (LHRH-a) or combination of chlormadinone acetate (CMA) and LHRH-a, and 19 patients with no treatments for prostate cancer were included in the analysis. Lumber spine bone mineral density (BMD) was measured by quantitative computed tomography. RESULTS: Aging had much influence on decreases of BMD than the other risk factors (p < 0.01). There were statistically decreases of BMD in the patients with CMA + LHRH-a compared with no treatments (p < 0.05). Adrenal androgen which had an important role of maintenance in BMD was statistically decreased by the administration of CMA (p < 0.01). CONCLUSIONS: Measurement of BMD before endocrine therapy is necessary for the patients with prostate cancer. It is important for the patients with decreases of BMD that CMA is not combined or the therapy for osteoporosis is preventively employed.

[Bone fracture receiving LH-RH agonists for prostatic cancer].

BACKGROUND: Luteinizing hormone-releasing hormone (LHRH) agonists are popularly used drugs in the treatment of prostatic cancer. However, it has been reported that continuation of a low testosterone level following a longterm administration of these drugs reduces the bone mineral density and makes for osteoporosis, which is accountable for fracture, we measured the bone mineral density and bone metabolic markers in the cases who suffered fracture receiving LHRH agonists for prostatic cancer. PATIENTS AND METHODS: Between 1994 and 1998, 196 patients (mean age 78.1 years) were treated with LHRH agonists for prostatic cancer. Of these patients, 13(7%) who had bone fracture during treated with LHRH agonists were divided into fracture group, and 70 patients who had not bone fracture divided into non-fracture group. Fracture by traffic accident was excluded. The bone density in the third lumbar vertebra was measured using quantitative computed tomography (QCT). Osteocalcin, 1, 25- (OH)2 vitamin D, urinary type 1 collagen cross-linked N-telopeptides (NTx), parathyroid hormone (PTH) and calcitonin were measured as bone metabolic markers. RESULTS: The mean age of fractured cases was 78 years. The period from the start of treatment to fracture was 11 to 45 months (mean 27 months). No case of fracture at the site of metastasis of prostatic cancer was found. The bone density was significantly low in the fracture group compared with that of non-fracture group. Of the bone metabolic markers, NTx showed high values in the fracture group. CONCLUSION: There is a need to measure bone mineral density and bone metabolic markers periodically and to evaluate secondary osteoporosis in the patients receiving LHRH agonists for prostatic cancer.

[Qualitative study of municipalities' maternal and child health promotion planning].

From 1997, basic maternal and child health services have been provided through municipalities. The ministry of Health and Welfare has requested all municipalities to publish a MCH promotion plan. We conducted a qualitative analysis of important contents of the plans using an evaluation index of original dichotomous variables. Out of 3,256 municipalities, 2,873 developed the plans for MCH promotion. Most of the plans present descriptions of the significance of MCH promotion planning, goals of the plans, problems of current MCH statistics and services, and plans of providing services. The proportion was low for plans which have needs assessment of target population, indicators of evaluation of plans, description of the relationship between objectives and services, importance of objectives, action plans, specific chapter or evaluation of the plan, monitoring of the plan, and plans for informing public of the MCH plan. Therefore, the MCH promotion plans have weaknesses in evaluation and action. The MCH promotion plans of middle sized cities were more likely to have these important contents. We will continue to analyze the processes for producing of excellent MCH promotion plans to extract universal promoting factors for producing MCH promotion plans.

[Papillary renal cell carcinoma complicated with emphysematous pyelonephritis: a case report].

A case of papillary renal cell carcinoma complicated with emphysematous pyelonephritis is reported. A 75-year-old woman with diabetes mellitus was admitted to our hospital with macroscopic hematuria and a child's head-sized firm mass in the left hypochondrium. Computed tomographic (CT) scanning revealed a huge left renal tumor with hydronephrosis. The patient had acute pyelonephritis and antibacterial therapy was initiated. Ten days later, conservative therapy was not effective and CT scan revealed emphysematous necrotic tissue in the tumor. We performed percutaneous drainage. Then dark red liquid and gas were discharged. Her general condition was improving. Left radical nephrectomy was performed. The mass was solid and pathological diagnosis was papillary renal cell carcinoma with necrotic tissue and coated by a thick pseudocapsule. She has remained free of disease for 27 months after operation.

[Percutaneous resection of renal pelvic fibroepithelial polyp with medullary sponge kidney: a case report].

Benign polyp of the renal pelvis is extremely rare. We report a case of fibroepithelial polyp in the renal pelvis complicated with medullary sponge kidney successfully treated by percutaneous resection. The patient had recurrent bilateral renal stones because of medullary sponge kidney. Percutaneous resection of renal pelvic polyps was carried out through a 26 Fr Amplatz sheath using a 24 Fr resectoscope. Pathological diagnosis was a fibroepithelial polyp. The etiology of this polyp was suggested to be chronic irritation of renal stone.

Multi-ubiquitination of a nascent membrane protein produced in a rabbit reticulocyte lysate.

During a large-scale in vitro translation analysis of a human full-length cDNA bank, we found many clones producing in vitro translation products showing ladder bands on a fluorogram with the equidistance of about 9 kDa at the position larger than the molecular mass expected from the open reading frame. We have analyzed a clone showing a typical pattern of the ladder bands. This clone encoded a 188-amino acid polypeptide containing a putative transmembrane domain. A green fluorescent protein-tagged polypeptide expressed in COS7 cells was localized in the endoplasmic reticulum and the Golgi apparatus. The ladder bands were observed in a rabbit reticulocyte lysate system, but not in a wheat germ extract system. Addition of the glutathione S-transferase-fused ubiquitin into the lysate caused upward shifts of the ladder bands. Addition of microsomal membranes prevented the formation of the ladder bands. Time course experiments demonstrated that the in vitro translation products increased in the presence of microsomal membranes, but were gradually degraded in their absence. These results suggest that the ladder formation resulted from the ubiquitination of misfolded polypeptide that failed to translocate to its proper position, and that an exclusion mechanism of misfolded membrane protein works in the rabbit reticulocyte lysate system.

[Double cancer observed from bladder cancer].

BACKGROUND: In recent years, despite of the improvement of treatment results for cancer and long life, the occurrence of second primary cancer was increased. In this paper, we analyzed present condition of double cancer observed with bladder cancer in our hospital. METHOD: Last 21 years, we have treated 969 cases (828 male and 141 female) of primary bladder cancer. For those cases, we analyzed in term of frequency, involved organ, age, interval between two cancer occurrence, risk factor and prognosis of double cancer patients. RESULT: Of 969 cases with bladder cancer, 81 cases (8.36%) had double cancer involving 6 cases (0.61%) of triple cancer. In sex, 70 males (9.78%) and 11 females (7.80%) had double cancer. As involved organs, 25 cases (3.02%) had in prostate, 23 cases (2.37%) in stomach, 3 case (2.13%) in breast, 14 cases (1.44%) in colon and rectum. In diagnosis timing of complicated cancer from bladder cancer, 28 cases (34.6%) were diagnosed previously to bladder, 28 cases (34.6%) were simultaneously and 31 cases (38.3%) were secondary. An average interval of diagnosis of two cancer were 49 +/- 42.5 months. An average age of occurrence of second cancer was 70.3 +/- 8.8 years. Actual survival rate from diagnosis of bladder cancer were 90.8%, 68.6%, 53.3% and 30.3%, after 1, 3, 5 and 10 years, respectively. Ten cases were dead by bladder cancer, 21 cases by complicated cancer and 16 cases by another cause. CONCLUSION: The incidence of double cancer with bladder cancer were increased. Prostate cancer, colorectal cancer and breast cancer were gradually increased as complicated organs in Japan. The prognosis of double cancer patients with bladder cancer was poor than single bladder cancer patients.

Cloning of human polyubiquitin cDNAs and a ubiquitin-binding assay involving its in vitro translation product.

During large-scale in vitro translation analysis of a human full-length cDNA bank, we found a clone producing a remarkably smaller translation product than that expected from the open reading frame. The cDNA encodes a polyubiquitin, UbC, composed of nine tandem repeats of the ubiquitin unit. The bank contained twelve UbC cDNAs including four full-length ones. Sequencing analysis of these clones showed that UbC cDNAs can be classified into two types, UbC1 and UbC2, in each of which there are six polymorphic nucleotide variations. The present UbC cDNA was in vitro translated in a rabbit reticulocyte or wheat germ extract to produce a free ubiquitin labeled with [35S]methionine. The labeled ubiquitin could be used as a substrate for thiol ester formation with ubiquitin-activating enzyme E1 or ubiquitin-conjugating enzyme E2.

[Stone recurrence after stone free status with extracorporeal shock wave lithotripsy].

BACKGROUND: The objects of this study is to evaluate the ipsilateral stone recurrence who became stone free status after extracorporeal shock wave lithotripsy (ESWL). METHODS: Three hundred ninety five patients who became stone free after ESWL with Lithostar and followed more than 6 months, were analyzed stone recurrence. Stone recurrence were diagnosed by KUB and/or ultrasound. Actual recurrence free rate were calculated based on the period from the day of achieved stone free status to the day of estimated recurrence. Eight factors examined included sex, side, number, location, size, stone history urological complication, hydronephrosis and also contralateral recurrence was analyzed. RESULTS: Over all ipsilateral recurrence free rate were 96.5%, 78.8%, 65.3% after 1, 3 and 5 years, respectively. Contralateral stone free rate were 98.1%, 92.5%, 87.2% after 1, 3 and 5 years. Five-year recurrence free rate according stone factors, there were significant difference in stone number (Single 71.1% and multiple 31.6%), in stone history (with history 77.1% and without 35.7%), in urological complication (without complication 67.7% and with complication 35.7%). However, there were no significant difference in sex, side, stone, location, stone size and hydronephrosis. CONCLUSION: This results suggested that the stone number, stone history and urological complication were highly related to ipsilateral stone recurrence after stone free status by ESWL. Extracorporeal shock was lithotripsy had probability of higher stone recurrence rate.

[Foreskin retraction for phimosis of the newborn].

BACKGROUND: No guideline exists on how to treat boy's phimosis. We examined if retraction of the foreskin of the newborn boy's penis could make true phimosis become false phimosis. METHODS: We taught the mother to retract the foreskin and keep inside the foreskin clean. Exposure degree of glans by retraction of foreskin was defined in 7 grades, 0 (none) approximately III (middle) approximately VI (full). RESULTS: Of the 538 newborn examined, none had full exposure (VI). All of the 372 cases who continued the procedure, including 2 buried penis, gained full exposure (VI). Average time for full exposure according to the first degree of exposure was 2.94 months (0), 1.78 months (III), 1.22 months (V), 2.32 months average, respectively. No serious complications occurred. CONCLUSION: Retraction of the foreskin from the newborn period made all the true phimosis to be false phimosis and operative procedures became unnecessary.

Stimulation by proopiomelanocortin-derived peptides of LH release by bullfrog dispersed anterior pituitary cells.

Labeling and immunoprecipitation experiments confirmed that three proopiomelanocortin (POMC)-derived peptides, the N-terminal peptide of POMC (NPP), joining peptide (JP) and adrenocorticotropic hormone (ACTH), were released by the bullfrog (Rana catesbeiana) anterior pituitary. The effects of these three peptides on luteinizing hormone (LH) release by bullfrog dispersed anterior pituitary cells were studied. NPP and ACTH, but not JP, enhanced LH release concentration-dependently. Approximately 6 hr elapsed before the gonadotrophs responded to NPP and ACTH by releasing LH, whereas their response to human GnRH (hGnRH) was faster, suggesting that the modes of action of these two peptides and hGnRH differ. These results raise the possibility that NPP and ACTH act as paracrine factors in the bullfrog pituitary to enhance LH release either directly or indirectly.

Contrasting patterns of expression of thyroid hormone and retinoid X receptor genes during hormonal manipulation of Xenopus tadpole tail regression in culture.

The precocious induction of amphibian metamorphosis is an ideal system for analyzing the developmental action of TH, while the hormonal activation of tadpole tail regression offers the further advantage of studying programmed cell death. One of the striking features of thyroid hormone (TH)-induced tail regression (as with morphogenetic responses of all tadpole tissues) in Xenopus is the rapid autoinduction of TRbeta gene, but it is not known how TH would affect the expression of the genes encoding TR's heterodimeric partner, retinoid X receptor (RXR). Here we first show that the synthetic glucocorticoid dexamethasone (Dex) potentiates and prolactin (PRL) suppresses, 3,3',5-triiodothyronine (T3)-induced regression of pre-metamorphic Xenopus tadpole tails in organ culture. T3 strongly upregulated (11-35-fold) the concentration of Xenopus TRbeta (xTRbeta) mRNA in these cultures while downregulating by 50% that of Xenopus RXRgamma (xRXRgamma) mRNA in the same samples of tail RNA. DEX and PRL enhanced or diminished the T3-regulated expression of these two transcripts, respectively, which parallels their other effects in whole tadpoles or cultured tails. The contrasting effects of the three hormones on the steady-state levels of xTRbeta and XRXRgamma mRNAs were time- and dose-dependent. T3 and DEX also strongly upregulated the transcription of xTRbeta gene transfected into Xenopus XTC-2 cells but PRL failed to prevent this autoinduction. The actions of these three hormones involved in amphibian metamorphosis, as judged by the expression of xTRbeta and xRXRgamma genes, reveal a new facet of hormonal interplay underlying their developmental actions.

[An analysis of factors related to recurrence of superficial bladder cancer after transurethral resection].

A total of 205 patients with primary superficial bladder cancer (Ta, T1) followed more than 3 years were retrospectively analyzed for factors related to recurrence of tumors after transurethral resection. Patients age were 25 to 90 years old, average 61 years old, and there were 160 males and 45 females. Initial tumor grades were G0 in 4 patients, G1 in 48, G2 in 134 and G3 in 19. Seventy four patients had Ta tumor and 131 had T1. Initial treatments were transurethral resection (TUR) alone in 137 patients. TUR with intravesical chemotherapy in 64, with BCG therapy in 7 and others in 7. Factors examined included age, sex, chief complaint, shape, size, and number of tumors, tumor distribution (single area or multiple area), histological grade, stage and intravesical chemotherapy. Overall non-recurrent rate were 81.7% at 1 year, 60.7% at 3 year, 53. 8% at 5 year and 44.2% at 8 year. Five-year non-recurrent rate according tumor factors, showed significant difference regarding tumor size (< 1 cm or 1 cm <: P = 0.027), tumor number (single or multiple: P = 0.004), tumor distribution (single area or multiple area: p = 0.002), histological grade (< G1 or G2 < : p = 0.001) and stage (Ta or T1: p = 0001). However, there were no significant difference regarding factors of age, sex, chief complaint, tumor figure and presence or absence of intravesical chemotherapy. This results suggested that the tumor factors of size, number, tumor distribution, grade and stage were highly related to intravesical tumor recurrence of superficial bladder cancer.

[A study of tumor location and prognosis in renal pelvic and ureteral cancer].

A total of 245 patients with renal pelvic and ureteral cancer (transitional cell carcinoma) were retrospectively analysed for tumor location and prognosis. In 133 renal pelvic cancer patients, 34 patients (25.6%) had tumor in lower calyx, 33 patients (24.8%) in renal pelvis, 31 patient (23.3%) in upper calyx, 21 patients (15.8%) in whole renal pelvis and 7 patients (5.2%) in middle calyx, respectively. In 128 ureteral cancer patients, 60 patients (46.9%) had tumor in lower ureter, 27 patients (21.1%) in distal end of ureter, 26 patients (20.3%) in middle ureter, 12 patients (9.4%) in upper ureter and 3 patients (2.3%) in whole ureter. In combination of tumor location, 101 patients (41.2%) had tumor in only renal pelvis, 94 patients (38.4%) had in only ureter, 14 patients (5.7%) had in renal pelvis and ureter, 19 patients (7.8%) had in renal pelvis and bladder, 12 patients (4.9%) had in ureter and bladder, and 5 patients (2%) had in renal pelvis, ureter and bladder. Five year survival rate of renal pelvic cancer according to tumor location were 55.9% in upper calyx tumor, 60.8% in middle calyx tumor, 63.8% in lower calyx tumor, 60.2% in renal pelvic tumor and 63.8% in PUJ tumor, respectively. There were no significant difference between those 5 groups. Five years survival rate of ureteral cancer according to tumor location, 90% in upper ureteral tumor, 60.8% in middle ureteral tumor, 66.5% in lower ureteral tumor and 52.6% in tumor of distal end of ureter, respectively. Also in those 4 groups, there were no significant difference.(ABSTRACT TRUNCATED AT 250 WORDS)

An additional arginine-vasotocin-related peptide, vasotocinyl-Gly-Lys, in Xenopus neurohypophysis.

The neurohypophysis of Xenopus and that of Ranidae and Bufonidae contain hydrin 1 (vasotocinyl-Gly-Lys-Arg) and hydrin 2 (vasotocinyl-Gly), respectively. In order to test the aldosterone-releasing activity of arginine vasotocin (AVT) and hydrin 1, purification of these peptides from an acid-extract of the neurointermediate lobe of Xenopus laevis was performed using an ODS-silica cartridge and reverse-phase and ion-exchange HPLC columns. As a result, an additional AVT-related peptide was newly found. Amino-acid analysis revealed that this peptide is vasotocinyl Gly-Lys (AVT-GK). The aldosterone-releasing activity of AVT-GK was equivalent to that of hydrin 1 (AVT-GKR) and lower than that of AVT. Like AVT and AVT-GKR, AVT-GK were effective in stimulating water flux from the isolated urinary bladder of the toad. Since AVT-GK is regarded as an intermediate between hydrin 1 and hydrin 2 in terms of its C-terminal form, it was designated hydrin 1'.

Purification and characterization of joining peptide and N-terminal peptide of proopiomelanocortin from the pars distalis of the bullfrog pituitary.

The joining peptide (JP) and the N-terminal peptide of proopiomelanocortin (NPP) were isolated from an acid-acetone extract of the distal lobe of the pituitary of the bullfrog, Rana catesbeiana, and purified by gel filtration and reverse-phase high performance liquid chromatography. The amino acid sequence of the bullfrog JP resembled the sequences of the JPs of Rana ridibunda (86% similarity) and Xenopus laevis (54% similarity), as deduced from the nucleotide sequences of their cDNAs. The amino acid sequence of bullfrog NPP showed 100%, 85%, and 50% similarity with those of Rana ridibunda, Xenopus laevis, and human NPPs, respectively. Administration of bullfrog NPP (0.05-5 micrograms/ml) to perifused Rana ridibunda interrenal slices induced a dose-dependent stimulation of corticosterone and aldosterone release. The present results indicate that the primary structure of NPP has been highly conserved during evolution. These data also reveal that NPP, which has no sequence homology with ACTH, exhibits a substantial corticotropic activity.