Development of an enzyme-linked immunosorbent assay system to determine the presence of antibodies specific for taxane structures.

Taxanes, which are widely used in treatment of numerous cancer types, are well-known to induce hypersensitivity reactions (HSR), especially in the case of paclitaxel. Although the cause of the HSR is commonly thought to be a non-immunological direct effect of the diluent which is used to dissolve paclitaxel, some reports suggest the possibility of the presence of an immunological reaction to the common taxane structure. The aim of this study was to establish a method to determine the presence of anti-taxane antibodies in body fluids of patients who have previously received paclitaxel, in order to estimate the risk of the occurrence of HSR to other taxane compounds, such as docetaxel. To prepare an enzyme-linked immunosorbent assay (ELISA) plate for determining taxanes, 10-deacetylbaccatin III (DAB) was first succinylated by use of dimethylaminopyridine and succinic anhydride in dried pyridine. After the succinylation reaction, three different products were obtained, and these were confirmed as 7-succinoyl DAB (7-DAB), 10-succinoyl DAB (10-DAB), and 7,10-disuccinoyl DAB (7,10-DAB) by (1)H-NMR analysis. Each of these three products was conjugated with bovine serum albumin (BSA), and adsorbed on an ELISA plate. By using a commercially available anti-taxane monoclonal antibody as a model antibody, the detection limit of the anti-taxane antibodies on the 7-DAB-BSA-, 10-DAB-BSA-, and 7,10-DAB-BSA-conjugated ELISA plate was estimated as 0.3, 1 and 10 ng/ml, respectively. The ELISA system established in this study may therefore be useful for estimating the risk of HSR to taxanes in a patient prior to the use of these drugs.

Mechanism of bladder dysfunction in idiopathic normal pressure hydrocephalus.

AIM: To elucidate the mechanism of bladder dysfunction in idiopathic normal pressure hydrocephalus (iNPH) by a urodynamic study. METHODS: Forty-two patients with possible iNPH, who were diagnosed by clinical symptoms/signs (gait, cognitive, and urinary disorders) with typical imaging features (ventricular enlargement) and normal cerebrospinal fluid pressure, were enrolled. The subjects included 36 men and 6 women; mean age, 72 years (62-83 years). All patients underwent a urodynamic test according to the definitions and methods proposed by the International Continence Society. RESULTS: Lower urinary tract symptoms were seen in 93% of the patients, with storage symptoms (93%) being more common than voiding symptoms (71%); and urinary urgency (overactive bladder) (64%)/frequency (64%) being more common than urinary incontinence (57%). The mean values for the maximum flow rate and post-void residual (PVR) volume were 11.7 ml/sec and 42.1 ml, respectively. PVR >100 ml was noted in six patients (three men, three women; range, 100-228 ml). Although the majority of patients had normal bladder volume at the first sensation (mean 134 ml), bladder capacity was small (mean 200 ml) and detrusor overactivity was seen in 95% of patients. CONCLUSIONS: While incontinence can result secondarily from gait disturbance or dementia, detrusor overactivity mostly underlies urinary urgency/frequency and incontinence in iNPH.

Development of the irradiation method for the first instar silkworm larvae using locally targeted heavy-ion microbeam.

To carry out the radio-microsurgery study using silkworm, Bombyx mori, we have already developed the specific irradiation systems for eggs and third to fifth instar larvae. In this study, a modified application consisting of the first instar silkworm larvae was further developed using heavy-ion microbeams. This system includes aluminum plates with holes specially designed to fix the first instar silkworm larvae during irradiation, and Mylar films were used to adjust energy deposited for planning radiation doses at certain depth. Using this system, the suppression of abnormal proliferation of epidermal cells in the knob mutant was examined. Following target irradiation of the knob-forming region at the first instar stage with 180-mum-diameter microbeam of 220 MeV carbon (12C) ions, larvae were reared to evaluate the effects of irradiation. The results indicated that the knob formation at the irradiated segment was specially suppressed in 5.9, 56.4, 66.7 and 73.6% of larvae irradiated with 120, 250, 400 and 600 Gy, respectively, but the other knob formations at the non-irradiated segments were not suppressed in either irradiation. Although some larva did not survive undesired non-targeted exposure, our present results indicate that this method would be useful to investigate the irradiation effect on a long developmental period of time. Moreover, our system could also be applied to other species by targeting tissues, or organs during development and metamorphosis in insect and animals.

Contribution of circulating hemocytes to the regeneration of heavy ion beams (12C5+) irradiated hematopoietic organs in the silkworm, Bombyx mori, through the way of phagocytosis of injured cells after invasion.

Heavy ion beam irradiation has promising effects on tumor therapy. Our previous study using the domesticated silkworm, Bombyx mori, showed that this irradiation could seriously damage larval hematopoietic organs but they would regenerate later. In the in vitro irradiation, most hemocytes died when hematopoietic organs and wing discs connected with epidermis were directionally irradiated from epidermis to hematopoietic organ and then cultured so as to exclude circulating hemocytes. A few hemocytes had escaped irradiation according to extremely low hematopoiesis in vitro. Almost no hemocytes could incorporate BrdU at 60 h after irradiation, with which living and proliferating hemocytes are also labeled. In the absence of circulating hemocytes, the irradiation-escaped hemocytes in the organs were not enough for cleaning all dead cells because lots of small dead bodies remained in situ post-irradiation. After irradiating hematopoietic organs in larvae (in vivo irradiation), only a few apoptotic cells were found when given the same length of recovery time, and most hemocytes maintained normal morphology. Many hemocytes incorporated BrdU when tested at the same time as the in vitro irradiation but this number was lower than that measured for control organs. Circulating hemocytes, labeled by fluorescent microbeads through phagocytosis before irradiation, were found to have invaded the in vivo irradiated hematopoietic organs where they help the irradiation-escaped hemocytes to clear dead cells in the process of regeneration. Hematopoiesis of the regenerated hematopoietic organs did not fully recover to the level of the control organs according to the number of hemocytes produced in tissue culture. Some of the released hemocytes obviously underwent apoptosis, suggesting a far-reaching bystander effect of carbon ion beams irradiation on hemocytes inside. From these results, it is suggested that, together with irradiation-escaped hemocytes, the invaded circulating hemocytes took part in the regeneration of heavy ion beams irradiated hematopoietic organs through the way of phagocytosis of injured hemocytes in vivo.

Probucol and atorvastatin decrease urinary 8-hydroxy-2'-deoxyguanosine in patients with diabetes and hypercholesterolemia.

To clarify whether probucol and statins suppress oxidative stress in diabetic patients, we studied the effects of probucol and the statin atorvastatin on urinary 8-hydroxy-2'deoxyguanosine (8-OHdG) levels in diabetics with hypercholesterolemia. A randomized, open study was performed on a total of 36 patients with type 2 diabetes and hypercholesterolemia. The patients were randomly assigned to a probucol group (500 mg/day, n = 18) or an atorvastatin group (10 mg/day, n = 18). During three months, total- and LDL-cholesterol decreased significantly in both groups. LDL-cholesterol was significantly lower in the atorvastatin group than probucol group. HDL-C decreased significantly in the probucol group and did not change in the atorvastatin group. 8-OHdG decreased significantly in both groups after 3 months; 12.4 +/- 7.5 to 8.1 +/- 4.2 ng/mg/Cr in the atorvastatin group (p < 0.05) and 12.3 +/- 8.8 to 6.8 +/- 2.6 ng/mg/Cr in the probucol group (p < 0.05), and these changes did not differ significantly between the two groups. But, in patients with high 8-OHdG levels (more than 10 ng/mg/Cr) before administration, urinary 8-OHdG decreased significantly from 19.5 +/- 4.9 to 9.2 +/- 3.4 ng/mg Cr (p < 0.01) in the atorvastatin group, and from 19.7 +/- 8.2 to 6.67 +/- 2.2 ng/mg Cr (p < 0.01) in the probucol group. Urinary 8-OHdG was significantly lower in the probucol group than in the atorvastatin group after the second and third months of administration (p < 0.05). These results suggest that while probucol and atorvastatin both reduce systemic oxidative stress, probucol might be the more useful in patients with strong oxidative stress.

Probucol delays progression of diabetic nephropathy.

Probucol has antioxidant and cholesterol-lowering effects. This study examined the effect of probucol on progression of diabetic nephropathy. We performed a randomized, open trial on 102 type 2 diabetes patients with clinical albuminuria (urinary albumin excretion >300 mg/g Cr). Fifty-one patients were assigned to probucol treatment (500 mg/day) and 51 to no probucol treatment. Among all patients, 40 who had serum creatinine >or=2mg/dl at baseline were defined as advanced cases. All patients were followed for a maximum 3 years. HbA1c levels were not different between two groups. High-density lipoprotein cholesterol decreased significantly in probucol group. Increase in urinary protein (g/day/month) was significantly greater in non-probucol than in probucol group. Hemodialysis was initiated in 23 patients (10 in probucol group and 13 in non-probucol group). The mean interval to initiation of hemodialysis was significantly longer in probucol group (20.7+/-8.2 months) than in non-probucol group (11.3+/-7.4 months). In advanced cases, increases of both serum creatinine and urinary protein were significantly suppressed in probucol group. In advanced cases, the hemodialysis-free rate was significantly higher in probucol group than in non-probucol group. These results suggest that probucol may suppress the progression of diabetic nephropathy.

Synthesis of silk fibroin-insulin bioconjugates and their characterization and activities in vivo.

The regenerated liquid silk fibroin with an average molecular mass of about 60 kDa consists of 18 kinds of amino acids containing approximately 10% of polar amino acids with hydroxyl and amino groups such as serine and lysine. The liquid silk fibroin is coupled covalently with insulin molecules through these strongly polar side groups by using glutaraldehyde. The physicochemical properties of the silk fibroin-insulin (SF-Ins) bioconjugates were investigated by enzyme-linked immunosorbent assay for the quantitative measurement of insulin. The biological activities of the insulin bioconjugates were characterized in vitro and in vivo. The SF-Ins constructs obtained by 5 h of covalent crosslinking showed much higher recovery (about 70%) and in vitro stability in human serum than bovine serum albumin-insulin (BSA-Ins) derivatives. The results in human serum indicated that the half-life in vitro of the biosynthesized SF-Ins derivatives was 2.1 and 1.7 times more than that of BSA-Ins conjugates and native insulin, respectively. The immunogenicity of the regenerated silk fibroin and the antigenicity of silk fibroin-modified insulin were not observed in both rabbits and rats. The pharmacological activity of the SF-Ins bioconjugates in diabetic rats evidently lengthened and was about 3.5 times as long as that of the native insulin, nearly 21 h. The bioconjugation of insulin with the regenerated silk fibroin greatly improved its physicochemical and biological stability.

[A case of interstitial pneumonia accompanying SIADH].

A 75-year-old man was transferred to our hospital on November 28, 2003 because of acute aggravation while being treated for interstitial pneumonia superimposed on pneumoconiosis at a local hospital. Upon admission, oxygen inhalation therapy and antimicrobial chemotherapy were started for the interstitial pneumonia. In addition, since he showed hyponatremia from admission, a saline load was administered and the clinical course was observed. However, disturbance of consciousness developed on January 5, 2004. At that time, the serum sodium was 115mEq/l. Since secretion of antidiuretic hormone (SIADH) had continued despite a low plasma osmolarity, we diagnosed syndrome of inappropriate secretion of antidiuretic hormone (SIADH). We initiated treatment with water restriction and saline load, but no remarkable improvement was observed. From February 7, 40mg /day prednisolone was started because of aggravation of interstitial pneumonia. As a result, the respiratory status and image findings improved, and serum sodium level was normalized. This case was considered to be SIADH secondary to interstitial pneumonia. Among respiratory tract diseases, SIADH is often caused by small cell lung carcinoma, although it may also occur concurrently with other respiratory tract diseases. Since hyponatremia may manifest grave disturbance of consciousness, investigation of the cause is important.

Severe hypercholesterolemia associated with decreased hepatic triglyceride lipase activity and pseudohyponatremia in patients after allogeneic stem cell transplantation.

A 55-year-old woman with Ph-negative acute lymphoblastic leukemia in primary induction failure received allogeneic peripheral blood stem cell transplantation from her HLA-compatible sister. Pseudohyponatremia developed due to extreme hypercholesterolemia of 4091 mg/dL accompanied by lipoprotein X and lipoprotein Y. The hypercholesterolemia was caused by cholestasis due to chronic GVHD and ischemic cholangiopathy. In addition, we found that hepatic triglyceride lipase (HTGL) activity was severely decreased, which could be another novel factor causing extreme hypercholesterolemia after allogeneic transplantation. The total cholesterol has been gradually decreasing followed by the improvement of cholestasis with bezafibrate, ursodeoxycholic acid and prednisone treatments, and by a slight increase in HTGL-protein. To our knowledge, this is the first report to describe the association of decreased HTGL with extreme hypercholesterolemia after allogeneic transplantation.

Synthesis, characterization and immunogenicity of silk fibroin-L-asparaginase bioconjugates.

L-asparaginase (ASNase) is one basic drug in the treatment of acute lymphoblastic leukemia (ALL). Because its half-life time is too short and it is easy to arouse allergic reaction, use in practical clinic is considerably limited. Silk fibroin (SF) with different molecular mass from 40 to 120 kDa is a natural biocompatible protein and could be used as a novel bioconjugate for enzyme modification to overcome its usual shortcomings mentioned above. When the enzyme was bioconjugated covalently with the water-soluble fibroin by glutaraldehyde, the enzyme kinetic properties and immune characteristics in vivo of the resulting silk fibroin-L-asparaginase (SF-ASNase) bioconjugates were investigated in detail. The results show that the modified ASNase was characterized by its higher residual activity (nearly 80%), increased heat and storage stability and resistance to trypsin digestion, and its longer half-life (63 h) than that of intact ASNase (33 h). The abilities of intact and modified ASNases to arouse allergic reaction are 2(4) and 2(1) antibody titers, respectively. Bioconjugation of silk fibroin significantly helps to reduce the immunogenicity and antigenicity of the enzyme. The apparent Michaelis constants of the modified ASNase (K(m(app))=0.844 x 10(-3)mol L(-1)) was approximately six times lower than that of enzyme alone, which suggests that the affinity of the enzyme to substrate l-asparagine elevated when bioconjugated covalently with silk fibroin. SF-ASNase bioconjugates could overcome the common shortcomings of the native form. Therefore, the modified ASNase coupled with silk fibroin has the potential values of being studied and developed as a new bioconjugate drug.

Chylomicronemia caused by lipoprotein lipase gene mutation related to a hyper-response of insulin secretion to glucose.

A 39-year-old man with lipoprotein lipase (LPL) deficiency (height 177.7 cm, body weight 67 kg, and body mass index 21.2 kg/m2) showed severe hypertriglyceridemia (2,032 mg/dl). LPL activity and concentration were markedly low in postheparin plasma. LPL gene analysis revealed a homozygous mutation, Asp204 --> Glu in exon 5. Fasting plasma glucose (81 mg/dl) and insulin (2.7 microU/ml) levels were normal. Plasma glucose pattern during oral glucose (75 g) tolerance test was normal, however 30 minutes after glucose-loading the insulin secretion unexpectedly increased to 89.4 microU/ml. These data suggested that chylomicronemia might be related to a hyper-response of insulin secretion to glucose without obesity.

Removal of Pb(II) from water using keratin colloidal solution obtained from wool.

The aim of this study is to investigate the use of keratin colloidal solution, which was obtained from wool, for the removal of Pb(II) from water. The addition of keratin colloidal solution (15 g L(-1), 0.30 mL) to a Pb(II) solution (1.0 mM, 0.90 mL, pH 5.0) resulted in the formation and precipitation of a Pb-keratin aggregate. Measurement of the Pb(II) and protein concentrations in the supernatant solution revealed that 88 and 99 % of the Pb(II) and keratin protein were removed from the solution, respectively. The maximum Pb(II) uptake capacity of keratin in the colloidal solution was 43.3 mg g(-1). In addition, the Pb-keratin aggregate was easily decomposed via the addition of nitric acid, which enabled the recovery of Pb(II). However, aggregation did not occur in solutions with Pb(II) concentrations below 0.10 mM. Therefore, we used a keratin colloidal solution encapsulated in a dialysis cellulose tube to remove Pb(II) from 0.10 mM solutions, which enabled the removal of 95 % of the Pb(II). From these results, we conclude that keratin colloidal solution is useful for the treatment of water polluted with Pb(II).

A hemolymph major anionic peptide, HemaP, motivates feeding behavior in the sweetpotato hornworm, Agrius convolvuli.

We recently identified a novel feeding-modulating peptide, hemolymph major anionic peptide (HemaP), designated Bommo-HemaP (B-HemaP), from hemolymph of the silkworm Bombyx mori. B-HemaP has a unique biological activity in modulating the regular frequency of feeding motivation, which is accompanied by increased foraging behaviors. To confirm the conservation of the HemaP-regulated feeding mechanism in lepidopteran species, we purified and sequenced two candidate peptides from the hemolymph of larvae of the sweet potato hornworm Agrius convolvuli. Unlike B. mori, A. convolvuli had two forms of HemaP, which were designated Agrco-HemaP-1 (A-HemaP-1) and Agrco-HemaP-2 (A-HemaP-2). The amino acid sequence of A-HemaP-2 was identical with that of A-HemaP-1, except for O-glycosylation on the fifth amino acid, threonine, within the N-terminal region. The amino acid sequence of A-HemaP-1/A-HemaP-2 had only 32% identity with B-HemaP. Structural analysis revealed that the carbohydrate moiety of A-HemaP-2 was an α-GalNAc residue. Injection of A-HemaP-1, A-HemaP-2 and recombinant A-HemaP-1 (rA-HemaP-1) individually caused a significant increase in foraging behaviors in A. convolvuli larvae, and no significant differences were observed among these three A-HemaPs. The CD spectra of these three A-HemaPs were quite similar, and all had α-helix-rich secondary structures. Although A-HemaP-1 and B-HemaP did not exhibit cross-reactivity at any injection doses examined, HemaP might be a conserved molecule among lepidopteran species that can modulate feeding motivation through the fluctuation of peptide levels in hemolymph.

Diagnosis and management of type I and type V hyperlipoproteinemia.

Both type I and type V hyperlipoproteinemia are characterized by severe hypertriglyceridemia due to an increase in chylomicrons. Type I hyperlipoproteinemia is caused by a decisive abnormality of the lipoprotein lipase (LPL)- apolipoprotein C-II system, whereas the cause of type V hyperlipoproteinemia is more complicated and more closely related to acquired environmental factors. Since the relationship of hypertriglyceridemia with atherosclerosis is not as clear as that of hypercholesterolemia, and since type I and V hyperlipoproteinemia are relatively rare, few guidelines for their diagnosis and treatment have been established; however, type I and V hyperlipoproteinemia are clinically important as underlying disorders of acute pancreatitis, and appropriate management is necessary to prevent or treat such complications. Against such a background, here we propose guidelines primarily concerning the diagnosis and management of type I and V hyperlipoproteinemia in Japanese.

Application of the powder of porous titanium carbide ceramics to a reusable adsorbent for environmental pollutants.

The aim of this study is to investigate the utilization of the powder of porous titanium carbide (TiC) ceramics as a novel adsorbent or a material for solid-phase extraction (SPE). The adsorption and elution of inorganic and organic pollutants, Pb(II), 2,4,6-trichlorophenol (TCP), perfluorooctane sulfonate (PFOS), and perfluorooctanoic acid (PFOA), to the material were evaluated. The cartridge packed with TiC ceramics powder was used for the extraction test of pollutants. The solution containing pollutants at 1.0 µg mL(-1) was passed through the TiC cartridge, and the substances were almost quantitatively removed. Furthermore, the pollutants retained in the cartridge were eluted with 3N HCl for Pb(II) and with methanol for organic pollutants. The recoveries of pollutants were over 80%. In addition, we used the TiC cartridge for the solid-phase extraction of water samples (500 mL each of the distilled water and the river water) by adding pollutants at determined concentrations. Every pollutant was adsorbed almost quantitatively, and eluted by 3N HCl or methanol. From these results, we concluded that the powder of porous TiC ceramics is a useful reusable adsorbent for the water cleanup and solid-phase extraction.

Central projections of the wing afferents in the hawkmoth, Agrius convolvuli.

Flight behaviors in various insect species are closely correlated with their mechanical and neuronal properties. Compared to locusts and flies which have been intensively studied, moths have "intermediate" properties in terms of the neurogenic muscle activations, power generation by indirect muscles, and two-winged-insect-like flapping behavior. Despite these unique characteristics, little is known about the neuronal mechanisms of flight control in moths. We investigated projections of the wing mechanosensory afferents in the central nervous system (CNS) of the hawkmoth, Agrius convolvuli, because the mechanosensory proprioceptive feedback has an essential role for flight control and would be presumably optimized for insect species. We conducted anterograde staining of nine afferent nerves from the fore- and hindwings. All of these afferents projected into the prothoracic, mesothoracic and metathoracic ganglia (TG1, 2 and 3) and had ascending fibers to the head ganglia. Prominent projection areas in the TG1-3 and suboesophageal ganglion (SOG) were common between the forewing, hindwing and contralateral forewing afferents, suggesting that information from different wings are converged at multiple levels presumably for coordinating wing flapping. On the other hand, differences of projections between the fore- and hindwing afferents were observed especially in projection areas of the tegulae in the TG1 and contralateral projections of the anterior forewing nerve in the TGs and SOG, which would reflect functional differences between corresponding mechanoreceptors on each wing. Afferents comprising groups of the campaniform sensilla at the wing bases had prominent ascending pathways to the SOG, resembling the head-neck motor system for gaze control in flies. Double staining of the wing afferents and flight or neck motoneurons also indicated potential connectivity between them. Our results suggest multiple roles of the wing proprioceptive feedback for flight and provide the anatomical basis for further understanding of neuronal mechanisms of the flight system in moths.

Utilization of silkworm cocoon waste as a sorbent for the removal of oil from water.

The aim of this study is to investigate the utilization of silkworm cocoon waste, such as pierced or stained cocoons, as a sorbent material for the removal of motor and vegetable oils from water. The oil-sorption capacity, rate and reusability of the material were evaluated. The results show the high sorption capacity of the silkworm cocoon waste sorbent (42-52 g(oil)/g(sorbent) for motor oil and 37-60 g(oil)/g(sorbent) for vegetable oil). The oil sorbed onto the material could be recovered by squeezing the sorbent, and the squeezed material showed an oil-sorption capacity over 15 g(oil)/g(sorbent). We concluded that the material shows a high performance as a low cost and environmental friendly sorbent for the removal of oil from water.