Crystallization and Melting Behavior of Poly(3‐hydroxybutyrate)‐Based Blends

Summary: Blends of high molecular weight poly(R‐3‐hydroxybutyrate) (PHB) ( = 352 000 g · mol^?1), comprising of either low molecular weight poly(R‐3‐hydroxybutyrate) (D‐PHB) ( = 3 900 g · mol^?1) or poly[(R‐3‐hydroxybutyrate)‐co‐(R‐3‐hydroxyvalerate)] (PHBV) ( = 238 000 g · mol^?1) with 12 mol‐% hydroxyvalerate (HV) content as a second constituent, were investigated along with the thermal properties and morphologies. After isothermal crystallization, a lowering of the melting temperature of PHB can be observed with increasing content of the second component in the blends. This behavior points towards miscibility of the constituents both in the liquid and the solid state. Crystallization kinetics was studied under isothermal and non‐isothermal conditions. The overall kinetics of isothermal crystallization was analyzed in terms of the Avrami equation. Only one crystallization peak is observed in all cases for the PHB/D‐PHB and PHB/PHBV blends under the conditions studied. This demonstrates co‐crystallization of the constituents. The addition of D‐PHB or PHBV to PHB reduces the rate of crystallization of the blends compared to that of neat PHB. The corresponding activation energies of crystallization also decrease with an increasing concentration of the second constituent. Non‐isothermal crystallization, carried out with different cooling rates held constant, is discussed in terms of a quasi‐isothermal approach. The corresponding rate constants as functions of reciprocal undercooling show Arrhenius‐like behavior in a certain range of temperatures. At sufficiently high undercooling, the rate constants of crystallization for the isothermal process exceed those reflecting non‐isothermal conditions, whereas in the limit of low undercoolings, the rate constants become similar. Ring‐banded morphologies are observed when PHB is in excess. When the respective second component is the major component, fibrous textures of the spherulites develop.

Polarized micrograph of PHB/PHBV 90/10.     

Biochemical engineering of the acyl side chain of sialic acids stimulates integrin-dependent adhesion of HL60 cells to fibronectin

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The physiological precursor of all sialic acids is N-acetyl- D-mannosamine. The N-acyl side chain of sialic acid can be modified by exposure of cells to synthetic N-acyl-modified D-mannosamines. In a new experimental approach cells were cultivated in the presence of N-propanoyl- D-mannosamine. This unnatural precursor of sialic acid is taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acid in vitro and in vivo. Here we report on the biological consequences of the incorporation of the unnatural N-propanoylneuraminic acid into glycoconjugates of HL60 cells. Biochemical engineering of the acyl side chain of neuraminic acids activates beta(1)-integrins (VLA4 or VLA5), resulting in an increased adhesion of HL60 cells to fibronectin.     

Temporal pattern of herpes simplex virus type 1 infection and cell death in the mouse brain stem: influence of guanosine nucleoside analogues

Levels of bystander death occurring in herpes simplex virus type 1 (HSV-1)-infected mouse brain stems were studied, as well as the extent to which bystander death is influenced by guanosine nucleoside analogue treatment. Consecutive sections from brain stems of HSV-1-infected mice were stained alternately for (i) viral infection and (ii) cell death (TUNEL assay). Virus antigen was detectable in brain stems on day 3 of infection, while TUNEL staining was comparatively lower. An increase in the extent of TUNEL staining was observed on day 4 of infection. Despite this increase, however, the ratio of TUNEL-stained to infection marker-stained tissue still indicated that the amount of TUNEL staining remained lower than infection staining at this time point. On days 5 and 6 of infection, TUNEL staining continued to increase and the TUNEL/infection marker ratio switched on day 6 in favour of excess TUNEL staining, which was observed in and around the foci of infection, suggesting bystander death. The excess TUNEL staining on day 6 of infection was further increased on treatment with antivirals. The significance and implications of these results are discussed with respect to the nature and mechanism of action of the TUNEL assay, dynamics of primary HSV-1 infection, immunological influences and potential effects of antiviral treatment. The potential problems of the TUNEL assay are considered in the context of viral infection and the TUNEL assay, in combination with infection marker staining, may potentially provide a model system for quantitative analysis of true bystander death during HSV infection in vivo.     

DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an ~465-kD catalytic subunit, DNA-PK_cs. Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PK_cs. Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.     

A new presenilin Alzheimer's disease case confirms the helical alignment of pathogenic mutations in transmembrane domain 5

In a case of familial early onset Alzheimer's disease, a mutation was detected in exon 7 of the presenilin 1 gene at codon 226 with a resultant amino acid change from leucine (CTC) to arginine (CGC) (L226R). This is a novel finding, yet is consistent with the previously reported mutations at codons 222, 229, 233 and 237 in transmembrane domain 5 which show a helical alignment of mutations in this domain. We conclude that the cause of Alzheimer's disease in this patient is an authentic PS1 gene abnormality responsible for the patient's early onset Alzheimer's disease.     

Evaluation of a resorbable, in situ setting bone substitute in a sheep model

The gold standard for bone substitution is the autologous bone graft, but because of its limited supply and the associated morbidity, the search for synthetic alternatives is necessary. A new in situ setting tricalcium phosphate cement was implanted in a trepanation defect (9.4 mm diameter, 10 mm depth) in the distal femoral epiphysis of sheep. Empty cavities and autologous bone graft were used as controls. Histologic and histomorphometric examinations were carried out after 12 weeks. Nearly 90% of the implanted cement was resorbed and replaced by ingrown bone with close contact between surrounding bone, new bone, and remaining cement particles. The amount of bone in the defect area was significantly higher in defects filled with cement relative to defects filled with autologous bone graft (mean 27 vs. 21%, 95% confidence intervals 23 to 31 and 18 to 23, p = 0.026). In conclusion, this new in situ setting cement is bioactive, resorbable, and osteoconductive. It will be useful as an alternative to autologous bone graft to fill stable defects.     

Rewarding effects of the optical isomers of 3,4-methylenedioxy-methylamphetamine ('Ecstasy') and 3,4-methylenedioxy-ethylamphetamine ('Eve') measured by conditioned place preference in rats

3,4-methylenedioxy-methylamphetamine (MDMA) (‘Ecstasy’) and its analogue 3,4-methylenedioxy-methylamphetamine (MDE) (‘Eve’) are well known illicit street drugs mainly abused by young people. In spite of the actual research going on, the classification of their abuse potential remains unclear. Since secondary reinforcers are the main factors responsible for craving and relapse, the aim of our study was to assess the potency of MDMA and MDE in a second order reinforcement paradigm, i.e. conditioned place preference (CPP). For the general assessment of our study conditions, we compared MDMA with amphetamine. Unexpectedly, no significant CPP for MDMA was found in contrast to amphetamine. Detailed analysis of current literature led us to the working hypothesis that social environment is crucial for the development of CPP. In a subsequent experiment we tested the influence of housing conditions on CPP using MDMA and demonstrated that isolated animals show significant CPP compared to group-housed ones. In order to better understand the rewarding mechanisms of Ecstasy-derivatives, we tested both the racemic drugs and the pure isomers in the CPP paradigm. Both MDMA's optical isomers and racemic MDMA showed significant CPP without notable differences, while MDE and its isomers completely failed to show any significant CPP. In conclusion, the mechanism by which MDMA induces addiction is much more complicated than assumed so far and more pronounced in isolated animals. The fact that both optical isomers of MDMA led to CPP implies that at least two pathways by which MDMA induces craving behaviour exist.     

Genomic gain of PIK3CA and increased expression of p110alpha are associated with progression of dysplasia into invasive squamous cell carcinoma

The regulation of apoptosis in atherosclerosis is not completely defined. The aim of this study was to determine the expression of Bcl-2, Bcl-x, Bax, and Bak in relation to apoptosis in advanced atherosclerotic lesions. In atherectomy (15), endarterectomy (10), and control non-atherosclerotic segments of renal (2) and of coronary and carotid (5) arteries, the extent of apoptosis was determined using TdT dUTP nick end labelling (TUNEL) and nuclear morphology (karyorrhexis/pyknosis) and expression of apoptosis regulators by immunohistochemistry and western blot analysis on paraffin-embedded material. In all specimens, the atherosclerotic involvement was advanced: grade V (n=18) and grade VI (n=7). The apoptotic index was high (mean 30%) in advanced lesions compared with controls (<2%) and smooth muscle cells (SMCs) were the predominant cell type undergoing apoptosis. In all TUNEL-positive apoptotic cells, Bax and Bak were present, while Bcl-x was absent. Bcl-2 was absent in a majority of these cells, but occasional TUNEL-positive cells expressed Bcl-2. In non-apoptotic cells, Bcl-x was present and western blot detected only the long isoform, Bcl-xL, from the plaques. In conclusion, increased Bax and Bak coupled with lack/paucity of Bcl-2 and Bcl-xL are associated with SMC apoptosis in advanced lesions. Bcl-xL in non-apoptotic cells appears to contribute to prolonged cell survival.