Retinoic Acid Deficiency Underlies the Etiology of Midfacial Defects.

Embryonic craniofacial development depends on the coordinated outgrowth and fusion of multiple facial primordia, which are populated with cranial neural crest cells and covered by the facial ectoderm. Any disturbance in these developmental events, their progenitor tissues, or signaling pathways can result in craniofacial deformities such as orofacial clefts, which are among the most common birth defects in humans. In the present study, we show that Rdh10 loss of function leads to a substantial reduction in retinoic acid (RA) signaling in the developing frontonasal process during early embryogenesis, which results in a variety of craniofacial anomalies, including midfacial cleft and ectopic chondrogenic nodules. Elevated apoptosis and perturbed cell proliferation in postmigratory cranial neural crest cells and a substantial reduction in Alx1 and Alx3 transcription in the developing frontonasal process were associated with midfacial cleft in Rdh10-deficient mice. More important, expanded Shh signaling in the ventral forebrain, as well as partial abrogation of midfacial defects in Rdh10 mutants via inhibition of Hh signaling, indicates that misregulation of Shh signaling underlies the pathogenesis of reduced RA signaling-associated midfacial defects. Taken together, these data illustrate the precise spatiotemporal function of Rdh10 and RA signaling during early embryogenesis and their importance in orchestrating molecular and cellular events essential for normal midfacial development.

Mucor pulmonary embolism in a patient with myelodysplastic syndrome.

Mucormycosis is a life-threatening infectious disease that occurs most commonly in immunocompromised patients such as those with hematological malignancies. Its clinical symptoms and associated radiological findings vary and specific biomarkers and culture characteristics have not been defined. An 85-year-old man who had been treated for myelodysplastic syndrome and tuberculosis for several months presented with subacute fever and worsening left-side chest pain. Contrast-enhanced computed tomography images depicted massive tumor-like consolidation without enhancement, expanding from the left lower lobe. Emboli that did not respond to anticoagulants were detected in the left descending pulmonary artery. Despite intensive treatment he developed multiple organ failure and died 47 days after hospitalization. Gross pathology of a lung autopsy specimen revealed left lower pulmonary arterial emboli and pulmonary infarction, which was concluded to be the direct cause of death. The emboli were histopathologically identified as invasive mycelia in vessels. Mucor sp. was detected via real-time polymerase chain reaction and immunohistopathological analyses revealed that the mold in the blood vessels of lung tissue was partially positive for the mucor antigen. In the present case of Mucor sp. pulmonary emboli in a patient with myelodysplastic syndrome, radiographic findings were hard to distinguish from those typical of a lung abscess.

Role of red blood cells in the anemia-associated bleeding under high shear conditions.

BACKGROUND: Red blood cells (RBCs) contribute to hemostasis under blood-flow, and anemia might contribute to a hemorrhagic diathesis. The majority of current laboratory techniques to assess hemostasis do not consider the effects of RBCs. An assay to determine the role of RBCs in hemostasis could be beneficial for clinical management. OBJECTIVES: To investigate the influence of RBCs in hemostasis. METHODS: Hemostasis was investigated using a novel microchip flow-chamber system (T-TAS® ) in an anemic patient with von Willebrand disease. Subsequently, the effects of RBCs in total thrombus analysis system (T-TAS) were examined using reconstituted whole blood at various hematocrit levels. RESULTS: In vivo: When the patient was anemic and demonstrated persisted hemorrhagic symptoms despite the maintained adequate von Willebrand factor ristocetin cofactor activity levels, thrombus formation determined by T-TAS was delayed. However, transfusions of RBCs resolved bleeding symptom and, accordingly, the thrombus formation in T-TAS improved. In vitro: Thrombus formation determined by T-TAS at 1000 s-1 was dose-dependent on hematocrit (the time to reach 10 kPa (T10 ): 10.0 ± 0, 9.5 ± 1.4, 6.7 ± 2.4, 2.8 ± 1.6 min at hematocrits of 0%, 12.5%, 25% and 50%, respectively). Markedly defective thrombus formation (T10 >10 min) was confirmed at a hematocrit <25% at 2000 s-1 . CONCLUSION: Red blood cells play an essential role in hemostasis under high shear, and RBC transfusions could be effective for refractory bleeding in patients with anemia. T-TAS measurements appear to reflect the hemostatic consequences of diminished red cell numbers under blood-flow, and could provide a valuable means for monitoring patients.

Assessing the clinical severity of type 1 von Willebrand disease patients with a microchip flow-chamber system.

BACKGROUND: The clinical phenotype of von Willebrand disease (VWD) is heterogeneous, and von Willebrand factor ristocetin cofactor activity (VWF:RCo) does not always reflect clinical severity, especially in VWD type 1. We have reported the potential of a microchip flow-chamber system (Total-Thrombus Formation Analysis System [T-TAS®]) for assessing physiologic hemostasis in VWD. Aim To evaluate the relationship between T-TAS, bleeding score (BS) and laboratory test results in type 1 VWD patients. METHODS: Microchips coated with collagen (platelet chip [PL-chip]) or collagen/thromboplastin (AR-chip) were used to assess platelet thrombus formation (PTF) at high shear rates or fibrin-rich PTF at low shear rates, respectively, in whole blood from 50 patients. The times needed for the flow pressure to increase by 10 kPa and 30 kPa (T10 and T30 ) from baseline were calculated from flow pressure curves. BS was determined by the use of a standardized questionnaire. RESULTS: PL-T10 values correlated with BS (R(2) ~ 0.45) better than VWF:RCo (R(2) ~ 0.36), irrespective of the flow rate, whereas AR-T10 showed only a weak correlation with BS (R(2) ~ 0.18). Patients with PL-T10 > 10 min or AR-T10 > 30 min had lower VWF levels and higher BS than those with PL-T10 ≤ 10 min or AR-T10 ≤ 30 min, and the greatest differences were observed with PL-T10. Clinical severity appeared to correlate best with PL-T10 > 8 min. BS was significantly higher in patients with VWF:RCo of < 10 IU dL(-1) than in those with VWF:RCo of 10 IU dL(-1) to < 25 IU dL(-1) and 25-40 IU dL(-1). In patients with VWF:RCo of < 10 IU dL(-1) , BS was significantly higher in those with PL-T10 > 8 min than in those with PL-T10 ≤ 8 min. CONCLUSION: T-TAS could be a useful technique for discriminating and predicting BS in VWD type 1 patients.

The pharmacokinetics and safety profiles of belimumab after single subcutaneous and intravenous doses in healthy Japanese volunteers.

WHAT IS KNOWN AND OBJECTIVES: Belimumab is a recombinant human monoclonal antibody that binds and antagonizes the biological activity of soluble B-lymphocyte stimulator (BLyS) protein. BLyS appears to play a role in the pathogenesis of systemic lupus erythematosus, and the biological profile of belimumab suggests that it may have a therapeutic benefit in the treatment for the disease. In this healthy Japanese subjects study, we investigated the pharmacokinetics and safety of a single subcutaneous and intravenous injection of belimumab administered as a 200 mg/mL liquid formulation. METHODS: This was an open-label, randomized, parallel-group, single-dose study in healthy Japanese subjects. Each subject received a single intravenous infusion or a subcutaneous injection of 200 mg belimumab. The pharmacokinetic parameters and safety parameters including local tolerance (injection site), biomarkers, immunogenicity and adverse events were evaluated up to 70 days post-dosing. RESULTS: After a single intravenous or a subcutaneous administration of 200 mg belimumab, all 16 subjects completed the study. There were no serious adverse events or adverse events related to injection site reactions. All seven adverse events were considered mild or moderate in intensity and deemed unrelated to belimumab except for cellulitis following intravenous administration. The bioavailability of the single subcutaneous dose of 200 mg belimumab in the subjects was estimated to be 77·5%. Time to the maximum serum concentration after subcutaneous injection was 6·5 days (median). The geometric mean terminal half-life was comparable between the two administration routes (17·7 days intravenous and 15·9 days subcutaneous). Serum immunoglobulin G level decreased slightly after each treatment. No subjects were found to produce antibelimumab antibodies. WHAT IS NEW AND CONCLUSIONS: A favourable absolute bioavailability in healthy Japanese subjects was seen following a subcutaneous injection of 200 mg belimumab. Considering the intersubject variability, exposures were consistent with those previously observed in healthy non-Japanese subjects. Safety and biomarker data were also consistent with previous non-Japanese clinical studies.

The pharmacokinetic and safety profiles of zanamivir after single and repeat intravenous administration in healthy Japanese males.

WHAT IS KNOWN AND OBJECTIVE: Neuraminidase inhibitors are important options for the treatment of infection by the influenza virus. For the treatment of severe influenza, parenteral administration of a neuraminidase inhibitor may be desirable. This study was conducted to evaluate the pharmacokinetic and safety profiles of intravenous zanamivir, an influenza viral neuraminidase inhibitor, in Japanese subjects to further characterize these profiles particularly following relatively high-doses when compared with inhalation doses and to provide reassurance that there are no marked differences with profiles reported for other ethnically different populations. METHODS: Single doses of 100, 300, 600 mg zanamivir were administered to healthy Japanese men in a randomized, double-blind, ascending dose, placebo-controlled, incomplete three-period cross-over study. In period 3, subjects were given 600 mg of zanamivir on day 1, followed by a 60 h washout period and then a 5-day course of 600 mg zanamivir twice daily. Each subjects received two of three active dosages of zanamivir from 100, 300 and 600 mg, and placebo. RESULTS: Adverse events reported in the study were all mild in intensity and resolved without any treatment. The mean AUC0-∞ values after single intravenous administration of 100, 300 and 600 mg were 16768, 53462 and 100400 ng·h/mL, respectively, demonstrating dose proportionality. No accumulation or time variance was observed after 5 days of twice-daily administration of 600 mg zanamivir. Urinary concentrations of zanamivir after single doses ranging from 100 to 600 mg indicated that over 94% of the zanamivir administered was excreted in urine within 24 h. WHAT IS NEW AND CONCLUSION: Single and 5-day BID repeat dosing of 600 mg were safely administered in Japanese healthy subjects. The pharmacokinetic profile of zanamivir after intravenous administration was consistent with previously reported findings in non-Japanese subjects.

Cloning, functional expression and promoter analysis of xylanase III gene from Trichoderma reesei.

In this study, the xyn3 gene from the filamentous mesophilic fungus Trichoderma reesei (Hypocrea jecorina) PC-3-7 was cloned and sequenced. Analysis of the deduced amino acid sequence of XYN III revealed considerable homology with xylanases belonging to glycoside hydrolase family 10. These results show that XYN III is distinguishable from XYN I and XYN II, two other T. reesei xylanases that belong to the glycosidase family 11. When xyn3 was expressed in Escherichia coli, significant activity was observed in the cell-free extract, and higher activity (13.2 U/ml medium) was recovered from the inclusion bodies in the cell debris. The sequence of the 5'-upstream region of the gene in the parent strain QM9414 is identical to that of PC-3-7, although the expression level of xyn3 in PC-3-7 has been reported to be at least 1,000 times greater than in QM9414. These results suggest that xyn3 expression in T. reesei QM9414 is silenced. The consensus sequences for ACEI, ACEII, CREI, and the Hap2/3/5 protein complex are all present in the upstream region of xyn3. Deletion analysis of the upstream region revealed that two regions containing consensus sequences for the known regulatory elements play important roles for xyn3 expression.

A comparison of vibration magnitudes on the tool with different subject according to the ISO 8662-7 standard.

Recently, ISO has defined the guidelines for the evaluation of the measurement and exposure limits of hand-transmitted vibrations at the handle of hand-held portable power tools. In a measurement of vibrations at pneumatic impact wrenches that tighten and remove screws. ISO 8862-7 standard: 1997 and ISO 5349 standard: 1986 are set as international standards. In order to make an exact measurement of the quantities of vibrations exposed to the hand-arm system of a human body, ISO 8662-7 gives specifications to a measurement device and a loading device. As to a measurement device, for instance, the mass, installation method etc. of transducers and mechanical filters are defined. In addition, it specifies with each tolerance the working conditions of a pneumatic tool, the number of revolutions of socket in a loading device, the feed force, the number of subjects, and such an evaluation method as how to decide a declaration value of vibrations. These specifications give toolmakers a comparative measurement of vibration values of their tools before shipment. Meanwhile, although the measurement condition described in ISO8662-7: 1997 is that skilled operators conduct vibration measurement, no provisions as to operators are made in detail. Hence, this study focuses its concern on ambiguous work experience years in measuring vibrations of the tools in conformity with ISO 8662-7, and hereby the effect of years of work experience upon measurement values is considered and at the same time a knowledge of how to select operators is made clear.

Quinone profiles of Thermoplasma acidophilum HO-62.

Quinones of Thermoplasma acidophilum HO-62 were analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. Menaquinone, methionaquinone, and 2-trans and 2-cis forms of thermoplasmaquinone were identified. The relative amount of thermoplasmaquinone increased under anaerobic conditions, and those of menaquinone and methionaquinone increased under aerobic conditions.

Occurrence of tetrodotoxin-related substances in the nontoxic puffer Takifugu xanthopterus.

Instead of tetrodotoxin, significant amounts of tetrodotoxin-related substances with no mouse lethality were detected in the nontoxic liver specimen of puffer fish, Takifugu xanthopterus. The tetrodotoxin-related substances, which were demonstrated to be tetrodotoxin derivatives by gas chromatography-mass spectrometry analysis, were similar to tetrodonic acid in HPLC but distinguishable from it in electrophoresis. Our results suggest that nontoxic puffer fish contains nontoxic tetrodotoxin derivatives as precursors or metabolites of tetrodotoxin.

Paralytic toxins in a ribbon worm Cephalothrix species (Nemertean) adherent to cultured oysters in Hiroshima Bay, Hiroshima Prefecture, Japan.

In 1998, during the surveillance of the toxicity of various marine fouling organisms in Hiroshima Bay, Hiroshima Prefecture, Japan, specimens of the ribbon worm, "himomushi" Cephalothrix sp. (Nemertean) adherent to the shells of cultured oysters hanging onto floating culture rafts were found to contain toxins which showed strong paralytic action in mice throughout the survey period, February to May. The maximum toxicity (as tetrodotoxin, TTX) was 14,734 MU/g whole body. Attempts were made to identify the paralytic toxins in this worm. The "himomushi" toxin (HMT) was extracted from the worm with 80% methanol acidified with acetic acid and the extract defatted with dichloromethane. The aqueous layer was chromatographed on activated charcoal and the unbound and bound toxic fractions were analyzed by high-performance liquid chromatography and gas chromatography-mass spectrometry. It was rather unexpectedly revealed from these results that HMT was comprised of TTX, 4-epiTTX, anhydroTTX and three unidentified toxins. To our knowledge, this is the first report of the occurrence of toxic organisms, containing a high concentration of TTX, adherent to cultured bivalves such as oysters.

Occurrence of a methyl derivative of saxitoxin in Bangladeshi freshwater puffers.

A new component of paralytic shellfish poison was isolated from a Bangladeshi freshwater puffer Tetraodon cutcutia. Its structure was deduced to be carbamoyl-N-methylsaxitoxin based on electrospray ionization mass spectrometry, [1H] NMR, and conversion experiments.

Photoaffinity labeling of the ligand-interacting helix of the retinoic acid receptor alpha.

Two photoaffinity-labeling probes for retinoic acid receptor (RAR) alpha, 4-[(3-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)carboxamido]ben zoic acid (3DIAM) and its para-isomer (4DIAM), were designed and synthesized. Both compounds possess high affinity for recombinant RAR alpha (MBP-RAR alpha/E) and bind covalently to its cognate ligand-binding site. The labeled site of MBP-RAR alpha/E with 3DIAM was determined, by the endoproteinase combination method, to be located in helix 11 of the ligand-binding domain of RAR alpha, which is the position at which the ligand is considered to bind, on the basis of the reported crystal structure of the retinoic acid/RAR gamma complex.

Design and synthesis of potent antitumor 5,4'-diaminoflavone derivatives based on metabolic considerations.

Recently, we reported that 5,4'-diaminoflavone (1) exhibits potent and specific growth-inhibitory activity against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7. However, when compound 1 was incubated with S-9 mix, its metabolites were observed. Moreover, addition of S-9 mix to the medium caused the drastic decrease in activity of compound 1. Since the 6-, 8-, and 3'-positions were considered to be metabolized oxidatively in vivo from MO calculations, a series of 5,4'-diaminoflavone derivatives substituted at such putative metabolic positions with various functional groups were synthesized aiming at the metabolically stable derivatives. Among them, 5,4'-diamino-6,8,3'-trifluoroflavone (14d) exhibited strong growth-inhibitory activity against MCF-7 cells even in the presence of S-9 mix. Moreover, orally administered compound 14d completely suppressed the growth of MCF-7 inoculated into nude mice, and the effect was more potent than that of compound 1. In addition to ER-positive breast cancer cells, compound 14d exhibited growth-inhibitory activity against a panel of human cancer cell lines including a part of ER-negative breast, endometrial, ovarian, and liver cancers. From these results, fluorine introduction to the putative metabolic positions of compound 1 was elucidated to be effective in the enhancement of the in vivo antitumor activity, probably due to the block of the metabolic deactivation.

Two new isomers of domoic acid from a red alga, Chondria armata.

Isodomoic acids G and H, two new isomers of the neurotoxin domoic acid, along with isodomoic acids A, B, E and F, were isolated from a red alga, Chondria armata, collected at the southern tip of Kyushu Island. The structures of two of these were deduced to be (E, E) and (Z, E) isomers of 2-carboxy-4-(5-carboxy-l-methyl-2-hexenylidene)-3-pyrro- lidineacetic acid, based on electrospray ionization mass and [1H]nuclear magnetic resonance spectral analyses including [1H-1H]correlation spectroscopy and nuclear Overhauser effect correlation spectroscopy.

Novel 5-aminoflavone derivatives as specific antitumor agents in breast cancer.

In the course of our search for new antitumor agents in breast cancer, novel amino-substituted flavone derivatives were synthesized and examined for antitumor activities. Among them, 5,4'-diaminoflavone and some of its congeners showed remarkable antiproliferative activity against the estrogen receptor (ER)-positive and estrogen-responsive human breast cancer cell line MCF-7. The activity was observed irrespective of the presence or absence of estrogen. The 5-aminoflavone derivatives (5-AFs) are not classical anti-estrogens because they did not compete with [3H]estradiol to bind the estrogen receptor. Moreover, 5-AFs showed antitumor activity highly selective to the ER-positive breast cancer cell line, and they showed no effects against the ER-negative human cancer cell lines HeLa S3, WiDr, and MDA-MB-453. Although the mechanism of their selective antitumor activity to ER-positive breast cancer cells is unclear, 5-AFs are expected to be a new type of antitumor agents in breast cancer.

A new saxitoxin analogue from a xanthid crab Atergatis floridus.

A novel paralytic toxin was isolated from toxic specimens of a xanthid crab Atergatis floridus inhabiting the Pacific coast of Shikoku Island. Its structure was deduced to be 11-saxitoxinethanoic acid (SEA) based on high-performance liquid chromatography, electrospray ionization mass spectrometry, and 1H and 13C-NMR spectrometries. This acid was assumed to exist as an equilibrium mixture of three tautomers, the main tautomer being the hydrate form 11 beta-epimer. SEA showed a specific toxicity of 830 mouse units per mumole on i.p. injection into mice.

Poisoning by the red alga 'ogonori' (Gracilaria verrucosa) on the Nojima Coast, Yokohama, Kanagawa Prefecture, Japan.

A food poisoning case due to the ingestion of 'ogonori', an edible red alga, occurred at Yokohama, Kanagawa Prefecture, Japan, in late October 1993, resulting in two victims, including one death (female). No causative agent present in the ogonori was found from a routine bioassay for marine toxins. From the production of increased amounts of prostaglandins (PGs), mainly PGE2, by the alga on stimulation by cutting or soaking in fresh water, and further increase of PGE2 by addition of arachidonic acid, it appeared that an enzyme, probably fatty acid cyclooxygenase, in the ogonori and the body of the victim, was acting on the highly unsaturated fatty acids in the oil of the ingested seafood and in the blood hemorrhaged from the stomach of the victim. This resulted in the production of over 30 mg of PGE2 and small amounts of other PGs in a comparatively short time. With this dosage the victim suffered from nausea, vomiting, and hypotension, and died of hypotensive shock. PGE2 seems to work more selectively on females. This type of poisoning is very unusual, and differs from the more familiar forms of poisoning occurring after ingestion of marine organisms.