Fatal tracheal aspergillosis during rituximab combined chemotherapy for diffuse large B-cell lymphoma that developed after lung transplantation.

Invasive tracheal aspergillosis (ITA) is an infection that is unique to patients who have undergone lung transplantation (LT). Although the activity of this disease often appears on imaging, we encountered a case of ITA that became exacerbated, despite few computed tomography (CT) findings, during rituximab combined chemotherapy for diffuse large B-cell lymphoma. ITA developed during immunosuppressive therapy after LT. Because CT findings may show false-negative results, bronchoscopy is recommended for such cases.

A locus for bipolar affective disorder on chromosome 4p.

The main clinical feature of bipolar affective disorder is a change of mood to depression or elation. Unipolar disorder, also termed major depressive disorder, describes the occurrence of depression alone without episodes of elevated mood. Little is understood about the underlying causes of these common and severe illnesses which have estimated lifetime prevalences in the region of 0.8% for bipolar and 6% for unipolar disorder. Strong support for a genetic aetiology is found in the familial nature of the condition, the increased concordance of monozygotic over dizygotic twins and adoption studies showing increased rates of illness in children of affected parents. However, linkage studies have met with mixed success. An initial report of linkage on the short arm of chromosome 11 (ref. 4) was revised and remains unreplicated. Reports proposing cosegregation of genes found on the X chromosome with bipolar illness have not been supported by others. More recently bipolar disorder has been reported to be linked with markers on chromosomes 18, 21, 16 and a region on the X chromosome different from those previously suggested. We have carried out a linkage study in twelve bipolar families. In a single family a genome search employing 193 markers indicated linkage on chromosome 4p where the marker D4S394 generated a two-point lod score of 4.1 under a dominant model of inheritance. Three point analyses with neighbouring markers gave a maximum lod score of 4.8. Eleven other bipolar families were typed using D4S394 and in all families combined there was evidence of linkage with heterogeneity with a maximum two-point lod score of 4.1 (theta = 0, alpha = 0.35).

Excellent performance of aromatic polyguanamines induced by multiple hydrogen bondable tetraazacalix[2]arene[2]-triazine ring in their main chain.

A series of poly(guanamine) (c-PG)s containing tetraazacalix[2]arene[2]-triazine (mPDA2CyC2) were successfully prepared by solution polycondensation of mPDA2CyC2 with various aromatic diamines in an aprotic organic solvent with a lithium chloride additive (5 wt%) at 150 °C for 6 hours. The number-average molecular weights (M n)s of these c-PG polymers reached up to 31 500, with a relatively broad molecular weight distribution (M w/M n) of 5.3. They showed good solubility in aprotic organic solvents, such as N-methylpyrrolidone and N,N-dimethylacetamide at a concentration of 2 mg mL-1. The glass transition temperatures (T g) of the c-PG polymers were in the range 359 °C-392 °C, approximately 160 °C higher than those of counterpart polymers (i.e., with no aza-calixarene-based PG (l-PG)). The coefficients of thermal expansion (CTEs) of the c-PG polymers were 29.7-48.1 ppm K-1 (at 100 °C-150 °C), much lower than those of l-PG samples, i.e., 59.1-85.1 ppm K-1. Transparent and almost colorless c-PG films were successfully prepared by a solution casting method, showing maximum tensile strength (σ S), modulus (E γ), and elongation at break (E b) values of 151 MPa, 6.3 GPa, and 4.4%, respectively, for the c-PG polymer from mPDA2CyC2 and 4,4'-oxydianiline monomers. The corresponding l-PG film exhibited σ S, E γ, and E b values of just 76 MPa, 5.4 GPa, and 1.6%, respectively. These outstanding thermal and mechanical properties of the c-PG polymers can be attributed to their multiple hydrogen bonding interaction between mPDA2CyC2 residues in the polymer backbone. This interaction was identified by infrared spectroscopy measurements at the broad absorption band around 3000-3400 cm-1.

Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells.

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).

Human diabetes associated with a mutation in the tyrosine kinase domain of the insulin receptor.

Insulin receptor complementary DNA has been cloned from an insulin-resistant individual whose receptors have impaired tyrosine protein kinase activity. One of this individual's alleles has a mutation in which valine is substituted for Gly996, the third glycine in the conserved Gly-X-Gly-X-X-Gly motif in the putative binding site fo adenosine triphosphate. Expression of the mutant receptor by transfection into Chinese hamster ovary cells confirmed that the mutation impairs tyrosine kinase activity.

A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons.

We tested the contribution of the small GTPase Rho and its downstream target p160ROCK during the early stages of axon formation in cultured cerebellar granule neurons. p160ROCK inhibition, presumably by reducing the stability of the cortical actin network, triggered immediate outgrowth of membrane ruffles and filopodia, followed by the generation of initial growth cone-ike membrane domains from which axonal processes arose. Furthermore, a potentiation in both the size and the motility of growth cones was evident, though the overall axon elongation rate remained stable. Conversely, overexpression of dominant active forms of Rho or ROCK was suggested to prevent initiation of axon outgrowth. Taken together, our data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics.

Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly.

Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.

Posttranslational cleavage of proinsulin is blocked by a point mutation in familial hyperproinsulinemia.

Familial hyperproinsulinemia is characterized by the accumulation of proinsulin-like material (PLM) in the plasma of affected patients. This disorder is inherited in an autosomal dominant fashion. The accumulation of PLM is thought to be due to the impaired conversion of proinsulin to insulin. Although PLM has been suggested to have an amino acid substitution, it has been impossible to locate and identify a substituted amino acid, due to the difficulty in isolating sufficient amounts of PLM from plasma samples. Therefore, we analyzed leukocyte DNA from one member of a proinsulinemic family, and we found a point mutation that changed guanine to adenine in the insulin gene. This transition implies that a substitution of histidine for arginine has occurred at amino acid position 65. Furthermore, it indicates that arginine at 65 is essential for the conversion of proinsulin to insulin. Our results suggest a novel mechanism by which disease can be incurred: a heritable disorder can result from a posttranslational processing abnormality caused by a point mutation.

Angiotensin II type 2 receptor overexpression activates the vascular kinin system and causes vasodilation.

Angiotensin II (Ang II) is a potent vasopressor peptide that interacts with 2 major receptor isoforms - AT1 and AT2. Although blood pressure is increased in AT2 knockout mice, the underlying mechanisms remain undefined because of the low levels of expression of AT2 in the vasculature. Here we overexpressed AT2 in vascular smooth muscle (VSM) cells in transgenic (TG) mice. Aortic AT1 was not affected by overexpression of AT2. Chronic infusion of Ang II into AT2-TG mice completely abolished the AT1-mediated pressor effect, which was blocked by inhibitors of bradykinin type 2 receptor (icatibant) and nitric oxide (NO) synthase (L-NAME). Aortic explants from TG mice showed greatly increased cGMP production and diminished Ang II-induced vascular constriction. Removal of endothelium or treatment with icatibant and L-NAME abolished these AT2-mediated effects. AT2 blocked the amiloride-sensitive Na(+)/H(+) exchanger, promoting intracellular acidosis in VSM cells and activating kininogenases. The resulting enhancement of aortic kinin formation in TG mice was not affected by removal of endothelium. Our results suggest that AT2 in aortic VSM cells stimulates the production of bradykinin, which stimulates the NO/cGMP system in a paracrine manner to promote vasodilation. Selective stimulation of AT2 in the presence of AT1 antagonists is predicted to have a beneficial clinical effect in controlling blood pressure.

Selective intracellular vaporisation of antibody-conjugated phase-change nano-droplets in vitro.

While chemotherapy is a major mode of cancer therapeutics, its efficacy is limited by systemic toxicities and drug resistance. Recent advances in nanomedicine provide the opportunity to reduce systemic toxicities. However, drug resistance remains a major challenge in cancer treatment research. Here we developed a nanomedicine composed of a phase-change nano-droplet (PCND) and an anti-cancer antibody (9E5), proposing the concept of ultrasound cancer therapy with intracellular vaporisation. PCND is a liquid perfluorocarbon nanoparticle with a liquid-gas phase that is transformable upon exposure to ultrasound. 9E5 is a monoclonal antibody targeting epiregulin (EREG). We found that 9E5-conjugated PCNDs are selectively internalised into targeted cancer cells and kill the cells dynamically by ultrasound-induced intracellular vaporisation. In vitro experiments show that 9E5-conjugated PCND targets 97.8% of high-EREG-expressing cancer cells and kills 57% of those targeted upon exposure to ultrasound. Furthermore, direct observation of the intracellular vaporisation process revealed the significant morphological alterations of cells and the release of intracellular contents.

Expression of the GLUT1 glucose transporter increases thymidine uptake in Chinese hamster ovary cells at low glucose concentrations.

An increase in expression of the GLUT1 glucose transporter gene has been observed to be associated with an increase in glucose transport activity upon oncogenic transformation of the cells. Increased expression of this glucose transporter isoform has been also observed in fetal tissues. To investigate the consequences of this phenomenon on cellular metabolism and cell growth, an expression vector containing the GLUT1 glucose transporter complementary DNA was transfected into Chinese hamster ovary cells. Overexpression of this glucose transporter isoform resulted in an increase in not only glucose uptake and utilization but also thymidine uptake when cells were exposed to glucose-deficient conditions. This increase in glucose metabolism and DNA synthesis may play an important role on the growth and/or survival of cancer cells and fetal tissues.

Mutations of a novel human RAD54 homologue, RAD54B, in primary cancer.

Association of breast tumor susceptibility gene products BRCA1 and BRCA2 with the RAD51 recombination protein suggested that cancer could arise through defects in recombination. The identification of NBS1, responsible for Nijmegen breakage syndrome, from the MRE11/RAD50 recombination protein complex also supports this hypothesis. However, our mutation analysis revealed that known members of the RAD52 epistasis group are rarely mutated in human primary cancer. Here we describe the isolation of a novel member of the SNF2 superfamily, characterized with sequence motifs similar to those in DNA and RNA helicases. The gene, designated RAD54B, is significantly homologous to the RAD54 recombination gene. The expression of RAD54B was high in testis and spleen, which are active in meiotic and mitotic recombination. These findings suggest that RAD54B may play an active role in recombination processes in concert with other members of the RAD52 epistasis group. RAD54B maps to human chromosome 8q21.3-q22 in a region associated with cancer-related chromosomal abnormalities. Homozygous mutations at highly conserved positions of RAD54B were observed in human primary lymphoma and colon cancer. These findings suggest that some cancers arise through alterations of the RAD54B function.

The effect of apo E secretion on lipoprotein uptake in transfected cells.

To investigate the role of apolipoprotein E (apo E) secreted by peripheral tissues in local lipoprotein metabolism, we developed a cell strain that constitutively produced and secreted apo E. A fusion plasmid containing rat apo E genomic DNA under control of mouse metallothionein promotor was constructed and transfected into Chinese hamster ovary cells. A stable transformant designated CHO-MAEII constitutively secreted rat apo E mainly in the form of sialylated free protein. The secretion was further enhanced by metal induction up to 1 micrograms apo E/ml per 12 h. When incubated with 125I-labeled very low density lipoprotein (125I-VLDL) at 37 degrees C, CHO-MAEII took up and degraded 125I-VLDL with higher affinity than control cells. Furthermore, considerable amount of methylated 125I-VLDL was degraded by CHO-MAEII, while no methylated 125I-VLDL was degraded by control cells. No significant differences were found in the uptake of 125I-LDL. The data indicated that apo E molecules secreted by CHO-MAEII were transferred to 125-VLDL particles, which caused a higher affinity of these particles for LDL receptors on the cells. It is suggested that apo E secreted from peripheral tissues enhances the uptake of lipoproteins by themselves or by surrounding cells in the local environment which demand cholesterol and express LDL receptors. CHO-MAEII was a good model for these 'auto- or paracrine-like functions' of apo E.

Increased liver glucose-transporter protein and mRNA in streptozocin-induced diabetic rats.

The effect of insulin-deficient diabetic states on the rat liver glucose-transporter (L-transporter isoform) protein and mRNA levels were studied. Rats were injected with 65 mg/kg streptozocin to induce diabetes and were maintained for 10 days and then treated with or without insulin for the next 5 days. The L-transporter isoform with apparent Mr of 55,000 was observed to be increased approximately twofold in the membranes from liver homogenates of diabetic rats compared with control rats when assessed by Western blot analysis with an anti-peptide antibody directed against rat L-transporter isoform. Insulin treatment of diabetic rats decreased the amount of L-transporter isoform protein toward levels observed in nondiabetic rats. Northern blot analysis demonstrated similar alterations in the rat L-transporter isoform mRNA that paralleled the changes observed in the L-transporter isoform protein. The increased levels of the L-transporter isoform protein and mRNA in diabetic rats are in marked contrast to the effects of insulin deficiency in rat adipocytes, which specifically decrease the amount of the adipocyte glucose-transporter isoform protein and mRNA. These results suggest that glucose-transporter isoforms in rat liver and adipocytes are regulated by different mechanisms and that an increased synthesis of the L-transporter isoform may contribute to the increased glucose output that occurs from the liver during insulin deficiency.

A functional retinoic acid receptor encoded by the gene on human chromosome 12.

A cDNA clone derived from a human hepatocellular carcinoma has been isolated on the basis of homology to the alpha human retinoic acid receptor (RAR alpha) gene. Expression of this cDNA produces a high affinity nuclear binding protein for retinoic acid. The product of this clone when expressed in transfected cells is able to activate transcription of a reporter plasmid through specific DNA sequences in response to the addition of retinoic acid to the medium. Dose-dependent profiles upon trans-activation of the reporter indicate that apparent sensitivity to retinoic acid of this protein is approximately 10-fold higher than that of human RAR alpha and is comparable to that of the second human RAR, RAR beta. This gene has been mapped to human chromosome 12, which is distinct from those coding for either alpha or beta RAR, and thus encodes a third human RAR.

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling.

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.

Atomoxetine reverses locomotor hyperactivity, impaired novel object recognition, and prepulse inhibition impairment in mice lacking pituitary adenylate cyclase-activating polypeptide.

Attention-deficit/hyperactivity disorder (ADHD) is a complex neurobehavioral disorder that is characterized by attention difficulties, impulsivity, and hyperactivity. A non-stimulant drug, atomoxetine (ATX), which is a selective noradrenaline reuptake inhibitor, is widely used for ADHD because it exhibits fewer adverse effects compared to conventional psychostimulants. However, little is known about the therapeutic mechanisms of ATX. ATX treatment significantly alleviated hyperactivity of pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP(-/-)) mice with C57BL/6J and 129S6/SvEvTac hybrid background. ATX also improved impaired novel object recognition memory and prepulse inhibition in PACAP(-/-) mice with CD1 background. The ATX-induced increases in extracellular noradrenaline and dopamine levels were significantly higher in the prefrontal cortex of PACAP(-/-) mice compared to wild-type mice with C57BL/6J and 129S6/SvEvTac hybrid background. These results suggest that ATX treatment-induced increases in central monoamine metabolism may be involved in the rescue of ADHD-related abnormalities in PACAP(-/-) mice. Our current study suggests that PACAP(-/-) mice are an ideal rodent model with predictive validity for the study of ADHD etiology and drug development. Additionally, the potential effects of differences in genetic background of PACAP(-/-) mice on behaviors are discussed.

Changes in cortical bone mineralization in the developing mandible: a three-dimensional quantitative computed tomography study.

Quantitative computed tomography (QCT) was completed in 34 subjects between the ages of 9 and 33 years with symmetrical mandibles in order to investigate the three-dimensional cortical bone mineral density (BMD) distribution in the mandible. The number and distribution of the pixels were determined at three levels: (1) representing the entire mandibular bone; (2) the cortical bone at 60% above the baseline defined as the segmentation level (around 1050 mg/cm3) and representative of only cortical bone; and (3) the highest mineralized cortical bone (>1250 mg/cm3). The geometrical distribution of the highest mineralized areas was evaluated by three-dimensional reconstruction of the images. The total number of pixels for the entire mandible increased significantly at each time point represented at four increasing ages groups (9-11 years of age, 12-14 years of age, 15-17 years of age, and >18 years of age). The male and female subjects had a similar total number of pixels for the entire mandible before the age of 11, but the male subjects showed a significantly larger total number of mandibular pixels after that age. Comparison of the number of pixels for pure cortical bone (60% segmentation level) and the highest mineralized cortical bone indicated a significant increase with maturation with the greatest change occurring between the 13-year and 16-year age groups. However, the ratio of cortical bone/total bone increased at a more rapid rate in the male subjects and reached a plateau by the 16-year age group, showing distinct differences in mineralization of the mandible between the sexes.

Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms.

Two cell lines have been established from insulinomas obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice. These cell lines, designated MIN6 and MIN7, produce insulin and T antigen and have morphological characteristics of pancreatic beta cells. MIN6 cells exhibit glucose-inducible insulin secretion comparable with cultured normal mouse islet cells, whereas MIN7 cells do not. Both cell lines produce liver-type glucose transporter (GT) mRNA at high level. Brain-type GT mRNA is also present at considerable level in MIN7 cells, but is barely detectable in MIN6 cells, suggesting that exclusive expression of the liver-type GT is related to glucose-inducible insulin secretion. MIN6 cells do not express either major histocompatibility (MHC) class I or class II antigens on the cell surface. However, treatment with interferon-gamma induces high levels of MHC class I antigens, and a combination of interferon-gamma and tumor necrosis factor-alpha induces a MHC class II antigen on the cell surface. These results emphasize that the MIN6 cell line retains physiological characteristics of normal beta cells. The MIN6 cell line will be especially useful to analyze the molecular mechanisms by which beta cells regulate insulin secretion in response to extracellular glucose concentrations. We discuss a possible role of GT isoforms in glucose sensing by beta cells.

Defect in tyrosine kinase activity of the insulin receptor from a patient with insulin resistance and acanthosis nigricans.

We report here a defect in tyrosine kinase activity of the insulin receptor from an insulin-resistant patient with acanthosis nigricans using cultured Ebstein-Barr virus (EBV)-transformed B-lymphocytes. As judged by affinity labeling and immunoblotting, the alpha- and beta-subunits of insulin receptors from the patient's lymphocytes exhibited the same mol wt as those from control subjects. Lectin-purified extracts from lymphocytes of the patient and the control subjects containing the same insulin-binding capacity were assayed for autophosphorylation and the ability to phosphorylate histone H2B. The degree of insulin-dependent autophosphorylation and the tyrosine kinase activity of the insulin receptor from the patient's lymphocytes were decreased to 15% and 13%, respectively, in a cell-free system. The insulin-dependent autophosphorylation of the insulin receptor was also impaired in intact EBV lymphocytes from the patient. Consistent with these results, we found that one of this patient's alleles had a mutation in which valine is substituted for Gly996, the third glycine in the conserved Gly-X-Gly-X-X-Gly motif in the kinase domain. Thus, it seems likely that the defect in tyrosine kinase activity of the insulin receptor cause the insulin resistance in this patient. The EBV lymphocyte can be a good system to detect genetically determined abnormalities in the insulin receptor.