Core-crosslinked compartmentalized cylinders

We present a detailed study on the preparation of compartmentalized cylindrical nanoparticles via a templated approach: the polybutadiene part of a linear polybutadiene-block-poly(2-vinyl pyridine)-block-poly(tert-butyl methacrylate) block terpolymer, B420V280T790, having a bulk microstructure with PB cylinders covered by a P2VP double helix and embedded in a PtBMA matrix was selectively crosslinked. Subsequent sonication-assisted dissolution and chemical modifications such as quaternization (P2VP to P2VPq) and ester hydrolysis (PtBMA to poly(sodium methacrylate), PMANa) resulted in core-crosslinked cylinders soluble in organic and aqueous media. Different amounts of crosslinker and the influence of the sonication treatment on size and shape of the cylindrical aggregates were investigated. The cylinders always exhibit a compartmentalized corona. Under certain conditions, in particular quaternization of P2VP in mixtures of THF and MeOH, the helical arrangement of the P2VPq shell could be preserved even in solution, whereas in most other cases randomly distributed P2VP/P2VPq patches were observed. In aqueous solution at high pH, intramicellar interpolyelectrolyte complex (im-IPEC) formation occurred between the positively charged P2VPq shell and the negatively charged PMANa corona. We further show that different noble metal nanoparticles can be generated either selectively within the im-IPEC compartments (Pd) or randomly distributed among shell and corona of the cylinders (Au and Pt).

Radical Chain Reduction of Alkylboron Compounds with Catechols

The conversion of alkylboranes to the corresponding alkanes is classically performed via protonolysis of alkylboranes. This simple reaction requires the use of severe reaction conditions, that is, treatment with a carboxylic acid at high temperature (>150 degrees C). We report here a mild radical procedure for the transformation of organoboranes to alkalies. 4-tert-Butylcatechol, a well-established radical inhibitor and antioxidant, is acting as a source of hydrogen atoms. An efficient chain reaction is observed due to the exceptional reactivity of phenoxyl radicals toward alkylboranes. The reaction has been applied to a wide range of organoboron derivatives such as B-alkylcatecholboranes, trialkylboranes, pinacolboronates, and alkylboronic acids. Furthermore, the so far elusive rate constants for the hydrogen transfer between secondary alkyl radical and catechol derivatives have been experimentally determined. Interestingly, they are less than 1 order of magnitude slower than that of tin hydride at 80 degrees C, making catechols particularly attractive for a wide range of transformations involving C-C bond formation.

Synthesis and Surface Modification of CdSe and CdS Quantum Dots Exhibiting High Quantum Yield

The thesis investigates the effect of surface treatment with various reducing and oxidizing agents on the quantum yield (QY) of CdSe and CdS quantum dots (QDs). The QDs, as synthesized by the organometallic method, contained defect sites on their surface that trapped photons and prevented their radiative recombination, therefore resulting in adecreased QY. To passivate these defect sites and enhance the QY, the QDs were treated with various reducing and oxidizing agents, including: sodium borohydride (NaBH4), calcium hydride (CaH2), hydrazine (N2H4), benzoyl peroxide (C14H10O4), and tert-butylhydroperoxide (C4H10O2). It was hypothesized that the reducing/oxidizing agents reduced the ligands on the QD surface, causing them to detach, thereby allowing oxygen from atmospheric air to bind to the exposed cadmium. This cadmium oxdide (CdO) layeraround the QD surface satisfied the defect sites and resulted in an increased QY. To correlate what effect the reducing and oxidizing agents were having on the optical properties of the QDs, we investigated these treatments on the following factors:chalcogenide (Se vs. S), ligand (oleylamine vs. OA), coordinating solvent (ODE vs.TOA), and dispersant solvent (chloroform vs. toluene) on the overall optical properties of the QDs. The QY of each sample was calculated before and after the various surface treatments from ultra-violet visible spectroscopy (UV-Vis) and fluorescence spectroscopy data to determine if the treatment was successful.From our results, we found that sodium borohydride was the most effective surface treatment, with 10 of the 12 treatments resulting in an increased QY. Hydrazine, on the other hand, was the least effective treatments, as it quenched the QD fluorescence in every case. From these observations, we hypothesize that the effectiveness of the QD surface treatments was dependent on reaction rate. More specifically, when the surface treatment reaction happened too quickly, we hypothesize that the QDs began to aggregate, resulting in a quenched fluorescence. Furthermore, we believe that the reactionrate is dependent on concentration of the reducing/oxidizing agents, solubility of the agents in each solvent, and reactivity of the agents with water. The quantum yield of the QDs can therefore be maximized by slowing the reaction rate of each surface treatment toa rate that allows for the proper passivation of defect sites.

Microwave-Assisted Bond Forming Reactions

ABSTRACT FOR PART I: PHOSPHA-MICHAEL ADDITIONS TO ACTIVATED INTERNAL ALKENES: STERIC AND ELECTRONIC EFFECTS A method for the phospha-Michael addition of bis(4-methyl)phenyl phosphine oxide to activated internal alkenes has been developed. Michael acceptors including cinnamates, crotonates, chalcones, and internal alkenes containing multiple activating groups were all successfully utilized in this reaction. The reaction was fairly tolerant of electron-donating and electron-withdrawing substituents on the Michael acceptor, and moderate to excellent yields (49-96%) of the adducts were isolated. When steric bulk was increased by a second substituent on the -position of the Michael-acceptor the reaction was suppressed. This was usually overcome by adding a second activating substituent to the -position. ABSTRACT FOR PART II: MICROWAVE-ASSISTED ARYLGOLD BOND FORMATION A microwave-assisted method was developed for the formation of arylgold complexes containing (2-Biphenyl)di-tert-butylphosphine (JohnPhos) as the supporting phosphine ligand. Arylboronic acids with increasingly bulky aromatic groups were screened to determine the steric limitations of the reaction. Arylgold complexes (JohnPhos)Au(p-methoxyphenyl), (JohnPhos)Au(2,4,6-trimethylphenyl), and (JohnPhos)Au(4-bromo-10-anthracene) were all synthesized by microwave irradiation at 70ºC in the presence of Cs2CO3 in either THF or iPrOH. Reactions performed with arylboronic acids containing unhindered ortho positions were carried out in THF. Arylboronic acids with substituents on the ortho position required iPrOH as the reaction solvent. Arylboronic acids with extreme steric hindrance on the ortho position of the aryl substituent, 2,4,6-triisopropylpphenylboronic acid, were unreactive. It was determined that increasing the irradiation temperature increased the formation of side products, therefore to promote conversion to the arylgold complex the duration of the reaction time was increased while maintaining a temperature of 70ºC. Arylgold complexes (JohnPhos)Au(p-methoxyphenyl), (JohnPhos)Au(2,4,6-trimethylphenyl), and (JohnPhos)Au(4-bromo-10-anthracene) were synthesized with moderate yields (40-69%).

Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome in which the known susceptibility genes (DKC1, TERC, and TERT) belong to the telomere maintenance pathway; patients with DC have very short telomeres. We used multicolor flow fluorescence in situ hybridization analysis of median telomere length in total blood leukocytes, granulocytes, lymphocytes, and several lymphocyte subsets to confirm the diagnosis of DC, distinguish patients with DC from unaffected family members, identify clinically silent DC carriers, and discriminate between patients with DC and those with other bone marrow failure disorders. We defined "very short" telomeres as below the first percentile measured among 400 healthy control subjects over the entire age range. Diagnostic sensitivity and specificity of very short telomeres for DC were more than 90% for total lymphocytes, CD45RA+/CD20- naive T cells, and CD20+ B cells. Granulocyte and total leukocyte assays were not specific; CD45RA- memory T cells and CD57+ NK/NKT were not sensitive. We observed very short telomeres in a clinically normal family member who subsequently developed DC. We propose adding leukocyte subset flow fluorescence in situ hybridization telomere length measurement to the evaluation of patients and families suspected to have DC, because the correct diagnosis will substantially affect patient management.

Genetic and pharmacologic manipulation of oxidative stress after neonatal hypoxia-ischemia

Oxidative stress is a critical component of the injury response to hypoxia-ischemia (HI) in the neonatal brain, and this response is unique and at times paradoxical to that seen in the mature brain. Previously, we showed that copper-zinc superoxide-dismutase (SOD1) over-expression is not beneficial to the neonatal mouse brain with HI injury, unlike the adult brain with ischemic injury. However, glutathione peroxidase 1 (GPx1) over-expression is protective to the neonatal mouse brain with HI injury. To further test the hypothesis that an adequate supply of GPx is critical to protection from HI injury, we crossed SOD1 over-expressing mice (hSOD-tg) with GPx1 over-expressing mice (hGPx-tg). Resulting litters contained wild-type (wt), hGPx-tg, hSOD-tg and hybrid hGPx-tg/hSOD-tg pups, which were subjected to HI at P7. Confirming previous results, the hGPx-tg mice had reduced injury compared to both Wt and hSOD-tg littermates. Neonatal mice over-expressing both GPx1 and SOD1 also had less injury compared to wt or hSOD-tg alone. A result of oxidative stress after neonatal HI is a decrease in the concentration of reduced (i.e. antioxidant-active) glutathione (GSH). In this study, we tested the effect of systemic administration of alpha-lipoic acid on levels of GSH in the cortex after HI. Although GSH levels were restored by 24h after HI, injury was not reduced compared to vehicle-treated mice. We also tested two other pharmacological approaches to reducing oxidative stress in hSOD-tg and wild-type littermates. Both the specific inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7NI), and the spin-trapping agent alpha-phenyl-tert-butyl-nitrone (PBN) did not reduce HI injury, however. Taken together, these results imply that H2O2 is a critical component of neonatal HI injury, and GPx1 plays an important role in the defense against this H2O2 and is thereby neuroprotective.

Cerebral vasculature is the major target of oxidative protein alterations in bacterial meningitis

We have previously shown that antioxidants such as a-phenyl-tert-butyl nitrone or N-acetylcysteine attenuate cortical neuronal injury in infant rats with bacterial meningitis, suggesting that oxidative alterations play an important role in this disease. However, the precise mechanism(s) by which antioxidants inhibit this injury remain(s) unclear. We therefore studied the extent and location of protein oxidation in the brain using various biochemical and immunochemical methods. In cortical parenchyma, a trend for increased protein carbonyls was not evident until 21 hours after infection and the activity of glutamine synthetase (another index of protein oxidation) remained unchanged. Consistent with these results, there was no evidence for oxidative alterations in the cortex by various immunohistochemical methods even in cortical lesions. In contrast, there was a marked increase in carbonyls, 4-hydroxynonenal protein adducts and manganese superoxide dismutase in the cerebral vasculature. Elevated lipid peroxidation was also observed in cerebrospinal fluid and occasionally in the hippocampus. All of these oxidative alterations were inhibited by treatment of infected animals with N-acetylcysteine or alpha-phenyl-tert-butyl nitrone. Because N-acetylcysteine does not readily cross the blood-brain barrier and has no effect on the loss of endogenous brain antioxidants, its neuroprotective effect is likely based on extraparenchymal action such as inhibition of vascular oxidative alterations.

Marked elevation in cortical urate and xanthine oxidoreductase activity in experimental bacterial meningitis

Experimental bacterial meningitis due to Streptococcus pneumoniae in infant rats was associated with a time-dependent increase in CSF and cortical urate that was approximately 30-fold elevated at 22 h after infection compared to baseline. This increase was mirrored by a 20-fold rise in cortical xanthine oxidoreductase activity. The relative proportion of the oxidant-producing xanthine oxidase to total activity did not increase, however. Blood plasma levels of urate also increased during infection, but part of this was as a consequence of dehydration, as reflected by elevated ascorbate concentrations in the plasma. Administration of the radical scavenger alpha-phenyl-tert-butyl nitrone, previously shown to be neuroprotective in the present model, did not significantly affect either xanthine dehydrogenase or xanthine oxidase activity, and increased even further cortical accumulation of urate. Treatment with the xanthine oxidoreductase inhibitor allopurinol inhibited CSF urate levels earlier than those in blood plasma, supporting the notion that urate was produced within the brain. However, this treatment did not prevent the loss of ascorbate and reduced glutathione in the cortex and CSF. Together with data from the literature, the results strongly suggest that xanthine oxidase is not a major cause of oxidative stress in bacterial meningitis and that urate formation due to induction of xanthine oxidoreductase in the brain may in fact represent a protective response.

Multiple pathways of neuroprotection against oxidative stress and excitotoxic injury in immature primary hippocampal neurons

In the immature brain hydrogen peroxide accumulates after excitotoxic hypoxia-ischemia and is neurotoxic. Immature hippocampal neurons were exposed to N-methyl-D-aspartate (NMDA), a glutamate agonist, and hydrogen peroxide (H(2)O(2)) and the effects of free radical scavenging and transition metal chelation on neurotoxicity were studied. alpha-Phenyl-N-tert.-butylnitrone (PBN), a known superoxide scavenger, attenuated both H(2)O(2) and NMDA mediated toxicity. Treatment with desferrioxamine (DFX), an iron chelator, at the time of exposure to H(2)O(2) was ineffective, but pretreatment was protective. DFX also protected against NMDA toxicity. TPEN, a metal chelator with higher affinities for a broad spectrum of transition metal ions, also protected against H(2)O(2) toxicity but was ineffective against NMDA induced toxicity. These data suggest that during exposure to free radical and glutamate agonists, the presence of iron and other free metal ions contribute to neuronal cell death. In the immature nervous system this neuronal injury can be attenuated by free radical scavengers and metal chelators.

Nitric oxide is protective in listeric meningoencephalitis of rats

The bacterium Listeria monocytogenes causes meningoencephalitis in humans. In rodents, listeriosis is associated with granulomatous lesions in the liver and the spleen, but not with meningoencephalitis. Here, infant rats were infected intracisternally to generate experimental listeric meningoencephalitis. Dose-dependent effects of intracisternal inoculation with L. monocytogenes on survival and activity were noted; 10(4) L. monocytogenes organisms induced a self-limiting brain infection. Bacteria invaded the basal meninges, chorioid plexus and ependyme, spread to subependymal tissue and hippocampus, and disappeared by day 7. This was paralleled by recruitment and subsequent disappearance of macrophages expressing inducible nitric oxide synthase (iNOS) and nitrotyrosine accumulation, an indication of nitric oxide (NO.) production. Treatment with the spin-trapping agent alpha-phenyl-tert-butyl nitrone (PBN) dramatically increased mortality and led to bacterial numbers in the brain 2 orders of magnitude higher than in control animals. Treatment with the selective iNOS inhibitor L-N(6)-(1-iminoethyl)-lysine (L-NIL) increased mortality to a similar extent and led to 1 order of magnitude higher bacterial counts in the brain, compared with controls. The numbers of bacteria that spread to the spleen and liver did not significantly differ among L-NIL-treated, PBN-treated, and control animals. Thus, the infant rat brain is able to mobilize powerful antilisterial mechanisms, and both reactive oxygen and NO. contribute to Listeria growth control.

TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.

Extracts of Lindera obtusiloba induce antifibrotic effects in hepatic stellate cells via suppression of a TGF-beta-mediated profibrotic gene expression pattern

Liver fibrosis is characterized by high expression of the key profibrogenic cytokine transforming growth factor (TGF)-beta and the natural tissue inhibitor of metalloproteinases (TIMP)-1, leading to substantial accumulation of extracellular matrix. Liver fibrosis originates from various chronic liver diseases, such as chronic viral hepatitis that, to date, cannot be treated sufficiently. Thus, novel therapeutics, for example, those derived from Oriental medicine, have gained growing attention. In Korea, extracts prepared from Lindera obtusiloba are used for centuries for treatment of inflammation, improvement of blood circulation and prevention of liver damage, but experimental evidence of their efficacy is lacking. We studied direct antifibrotic effects in activated hepatic stellate cells (HSCs), the main target cell in the fibrotic liver. L. obtusiloba extract (135 mug/ml) reduced the de novo DNA synthesis of activated rat and human HSCs by about 90%, which was not accompanied by cytotoxicity of HSC, primary hepatocytes and HepG2 cells, pointing to induction of cellular quiescence. As determined by quantitative polymerase chain reaction, simultaneous treatment of HSCs with TGF-beta and L. obtusiloba extract resulted in reduction of TIMP-1 expression to baseline level, disruption of the autocrine loop of TGF-beta autoinduction and increased expression of fibrolytic matrix metalloproteinase (MMP)-3. In addition, L. obtusiloba reduced gelatinolytic activity of HSC by interfering with profibrogenic MMP-2 activity. Since L. obtusiloba extract prevented intracellular oxidative stress experimentally induced by tert-butylhydroperoxide, we concluded that the direct antifibrotic effect of L. obtusiloba extract might be mediated by antioxidant activity. Thus, L. obtusiloba, traditionally used in Oriental medicine, may complement treatment of chronic liver disease.

Characterization of calcium signaling mechanisms in osteoblasts

Bone remodeling is controlled by the osteoclast, which resorbs bone, and the osteoblast, which synthesizes and secretes proteins that are eventually mineralized into bone. Ca$\sp{2+}$ homeostasis and signaling contribute to the function of nearly all cell types, and understanding both in the osteoblast is of importance given its secretory properties and interaction with osteoclasts. This study was undertaken to identify and investigate the physiology of the Ca$\sp{2+}$ signaling mechanisms present in osteoblasts. The Ca$\sp{2+}$ pumps, stores and channels present in osteoblasts were studied. RT-PCR cloning revealed that osteoblast-like cells express PMCA1b, an alternatively spliced transcript of the plasma membrane Ca$\sp{2+}$-ATPase. The PMCA1b isoform contains a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. The regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca$\sp{2+}$-ATPase.^ Calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, and the compartmentalization and regulated release of calcium is cell-type specific. Fura-2 was employed to monitor intracellular Ca$\sp{2+}$. Thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum Ca$\sp{2+}$-ATPase activity, both emptied a single intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The Ca$\sp{2+}$ storage system present in osteoblasts is typical of a non-excitable cell type, despite these cells sharing characteristics of excitable cells such as voltage-sensitive Ca$\sp{2+}$ channels (VSCCs).^ VSCCs are important cell surface regulators of membrane permeability to Ca$\sp{2+}$. In non-excitable cells VSCCs act as cellular transducers of stimulus-secretion coupling, activators of intracellular proteins, and in control of cell growth and differentiation. Functional VSCCs have been shown to exist in osteoblasts, however, no molecular cloning has been reported. To obtain information concerning the molecular identity of the osteoblastic VSCC, we used an RT-PCR regional amplification approach. Sequencing of the products indicated that osteoblasts express at least two isoforms of the L-type VSCC, $\alpha 1\sb{\rm C-a}$ and the $\alpha 1\sb{\rm C-d}$, which share regions of identity to the $\alpha \sb{\rm 1C}$ isoform first identified in cardiac myocytes. The ability of $1,25(\rm OH)\sb2D\sb3$ and structural analogs to modulate expression of Ca$\sp{2+}$ channel mRNA was then investigated. Cells were cultured for 48 hr in the presence of $1,25(\rm OH)\sb2D\sb3$ or vitamin D analogs, and the levels of mRNA encoding VSCC $\alpha \sb{\rm 1C}$ were quantitated using a competitive RT-PCR assay. It was found that $1,25(\rm OH)\sb2D\sb3$ and analog BT reduced steady state levels of $\alpha \sb{\rm 1C}$ mRNA. Conversely, analog AT did not alter steady state levels of Ca$\sp{2+}$ channel mRNA. Since it has been shown previously that analog BT, but not AT, binds and activates the nuclear vitamin D receptor, these findings suggest that the down regulation of channel mRNA involves the nuclear receptor for $1,25(\rm OH)\sb2D\sb3$. ^

Reactive oxygen intermediates contribute to necrotic and apoptotic neuronal injury in an infant rat model of bacterial meningitis due to group B streptococci.

Reactive oxygen intermediates (ROI) contribute to neuronal injury in cerebral ischemia and trauma. In this study we explored the role of ROI in bacterial meningitis. Meningitis caused by group B streptococci in infant rats led to two distinct forms of neuronal injury, areas of necrosis in the cortex and neuronal loss in the dentate gyrus of the hippocampus, the latter showing evidence for apoptosis. Staining of brain sections with diaminobenzidine after perfusion with manganese buffer and measurement of lipid peroxidation products in brain homogenates both provided evidence that meningitis led to the generation of ROI. Treatment with the radical scavenger alpha-phenyl-tert-butyl nitrone (PBN) (100 mg/kg q8h i.p.) beginning at the time of infection completely abolished ROI detection and the increase in lipidperoxidation. Cerebral cortical perfusion was reduced in animals with meningitis to 37.5+/-21.0% of uninfected controls (P < 0.05), and PBN restored cortical perfusion to 72.0+/-8.1% of controls (P < 0.05 vs meningitis). PBN also completely prevented neuronal injury in the cortex and hippocampus, when started at the time of infection (P < 0.02), and significantly reduced both forms of injury, when started 18 h after infection together with antibiotics (P < 0.004 for cortex and P < 0.001 for hippocampus). These data indicate that the generation of ROI is a major contributor to cerebral ischemia and necrotic and apoptotic neuronal injury in this model of neonatal meningitis.

Mononuclear Tetraamineplatinum(II) Complexes: Synthesis, Anticancer Activity, DNA Binding, and Cellular Uptake

The synthesis of three bis[(tert-butoxy)carbonyl]-protected (tetramine)dichloroplatinum complexes 2a – c of formula cis-[PtCl2(LL)] and of their cationic deprotected analogs 3a – c and their evaluation with respect to in vitro cytotoxicity, intramolecular stability, DNA binding, and cellular uptake is reported. The synthesis comprises the complexation of K2[PtCl4] with di-N-protected tetramines 1a – c to give 2a – c and subsequent acidolysis, yielding 3a – c. The cytotoxicity of the complexes is in direct relation to the length of the polyamine. Complexes 3a – c display a significant higher affinity for CT DNA as well as for cellular DNA in A2780 cells than cisplatin.