Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Stomatal malfunctioning under low VPD conditions: induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling?

Exposing plants to low VPD reduces leaf capacity to maintain adequate water status thereafter. To find the impact of VPD on functioning of stomata, stomatal morphology and leaf anatomy, fava bean plants were grown at low (L, 0.23 kPa) or moderate (M, 1.17 kPa) VPDs and some plants that developed their leaves at moderate VPD were then transferred for 4 days to low VPD (M→L). Part of the M→L‐plants were sprayed with ABA (abscisic acid) during exposure to L. L‐plants showed bigger stomata, larger pore area, thinner leaves and less spongy cells compared with M‐plants. Stomatal morphology (except aperture) and leaf anatomy of the M→L‐plants were almost similar to the M‐plants, while their transpiration rate and stomatal conductance were identical to that of L‐plants. The stomatal response to ABA was lost in L‐plants, but also after 1‐day exposure of M‐plants to low VPD. The level of foliar ABA sharply decreased within 1‐day exposure to L, while the level of ABA‐GE (ABA‐glucose ester) was not affected. Spraying ABA during the exposure to L prevented loss of stomatal closing response thereafter. The effect of low VPD was largely depending on exposure time: the stomatal responsiveness to ABA was lost after 1‐day exposure to low VPD, while the responsiveness to desiccation was gradually lost during 4‐day exposure to low VPD. Leaf anatomical and stomatal morphological alterations due to low VPD were not the main cause of loss of stomatal closure response to closing stimuli.     

L1 cell adhesion molecules as regulators of tumor cell invasiveness

Fast growing malignant cancers represent a major therapeutic challenge. Basic cancer research has concentrated efforts to determine the mechanisms underlying cancer initiation and progression and reveal candidate targets for future therapeutic treatment of cancer patients. With known roles in fundamental processes required for proper development and function of the nervous system, L1-CAMs have been recently identified as key players in cancer biology. In particular L1 has been implicated in cancer invasiveness and metastasis, and has been pursued as a powerful prognostic factor, indicating poor outcome for patients. Interestingly, L1 has been shown to be important for the survival of cancer stem cells, which are thought to be the source of cancer recurrence. The newly recognized roles for L1CAMs in cancer prompt a search for alternative therapeutic approaches. Despite the promising advances in cancer basic research, a better understanding of the molecular mechanisms dictating L1-mediated signaling is needed for the development of effective therapeutic treatment for cancer patients.Key words: L1CAMs, cancer, metastasis, axon guidance, cancer stem cell, migration, invasionA major obstacle in oncology is the early diagnosis and curative therapeutic intervention of locally invasive cancers that rapidly disseminate from the primary tumor to form metastases. The standard treatment for malignant tumors consists of surgical removal of the tumor mass followed by chemo- and radiotherapy in order to eradicate the remaining cancer cells. Despite such aggressive intervention, a population of resistant cancer cells often remains intact and is thought to be the source of cancer recurrence.During the past decades, cancer basic research has focused on determining the molecular mechanisms underlying cancer initiation and progression that can provide a basis for the development of new and effective therapeutic treatments for cancer patients. An important finding was the discovery that cancer onset and development are often associated with alterations in the expression of cell adhesion molecules, which are likely to stimulate tumor cell invasiveness by signaling mechanisms that enhance cell migration.¹ The L1 family of neural cell adhesion molecules (L1-CAMs), which is comprised of four structurally related transmembrane proteins L1, CHL1, NrCAM and neurofascin (Fig. 1), is now in the spotlight of cancer research due to their upregulation in certain human tumors. L1-CAMs are transmembrane molecules of the immunoglobulin superfamily, characterized by an extracellular region of six immunoglobulin-like domains and four to five fibronectin type III repeats, followed by a highly conserved cytoplasmic domain, which is reversibly linked to the cell cytoskeleton through binding to ankyrin and ERM proteins (ezrin-radixin-moesin).² Its multi-domain structure allows complex heterophilic interactions with diverse cell receptors, although homophilic interactions also have a crucial role in L1-CAMs mediated signaling.Open in a separate windowFigure 1L1-CAMs: All have 6 Ig domains and 4–5 FN domains. The 186 kD Neurofascin isoform has a mucin-like Pro/Ala/Thr-rich (PAT) domain, while the 155 kD has only the 4 FN domains. RGD and DGEA motifs interact with integrins, while the FigQ/AY motif binds to ankyrin. ERM binding sites are indicated. The RSLE motif in L1 recruits AP2/clathrin adaptor for endocytosis.A wealth of studies has revealed L1-CAMs as pivotal components for proper development of the nervous system through regulation of cell-cell interactions. L1-CAMs have critical roles in neuronal migration and survival, axon outgrowth and fasciculation, synaptic plasticity and regeneration after trauma.² Neither CHL1 nor L1 is present on mature astrocytes, oligodendroglia or endothelial blood vessel cells in the brain, but CHL1 is upregulated in astrocytes upon injury³ and is present on oligodendroglial precursors.^4,5 During neural development, L1 plays an important role in the migration of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.⁶ In the cerebellum, L1 is required for the inward migration of granule neurons from the external granular layer and cooperates with NrCAM in regulating neuronal positioning.² Similarly, CHL1 controls area-specific migration and positioning of deep layer cortical neurons in the neocortex.⁷ In addition to its role in neuronal precursor positioning, L1 plays a crucial role in axon guidance, which is governed by repellent and attractive response mechanisms directed by Ephrins and Semaphorins and their receptors (Ephs, Neuropilins, Plexins).² The importance of L1-CAMs in the development and function of the nervous system is exemplified by developmental neuropsychiatric disorders that are associated with mutation or genetic polymorphisms in genes encoding L1 (X-linked mental retardation) and CHL1 (low IQ, speech and motor delay). Polymorphisms in L1 and CHL1 genes are also associated with schizophrenia, and NrCAM gene polymorphisms are linked to autism in some populations.²Recent studies have described upregulation of L1 in a variety of tumor types. Overexpression of L1 correlates with tumor progression and metastasis in certain human gliomas,⁸ melanoma,⁹ ovarian¹⁰ and colon carcinomas.^11–13 Interestingly, L1 was found to be present only in cells at the invasive front of colon cancers but not in the tumor mass.¹² L1 is also associated with micrometastasis to both lymph nodes and bone marrow in patients bearing other cancers, suggesting a potential role in early metastatic spread.¹¹ L1 has now been pursued as both a biomarker and a powerful prognostic factor, indicative of poor outcome for patients as observed for epithelial ovarian carcinoma¹⁰ and colorectal cancer.¹¹ More recently, L1 has been shown to be overexpressed in a small fraction of glioma cells, termed glioma stem cells, which are capable of self-renewal and generate the diverse cells that comprise the tumor.¹⁴ First characterized in acute myeloid leukemia,¹⁵ cancer stem cells have been recently described in a variety of solid tumors, including breast cancer, lung cancer and gastrointestinal tumors.¹⁶ In gliomas, L1 expression was shown to be required for maintaining the growth and survival of glioma stem cells.¹⁴ These findings suggest that L1 may be implicated not only in cancer invasiveness but also in cancer survival. It will be important to determine if L1 is also upregulated in other cancer stem cells as well as to define the role of L1-mediated signaling in other cancers. Although not extensively investigated, NrCAM has also been shown to be overexpressed in glioblastoma cell lines and several cases of high grade astrocytoma¹⁷ and ependymomas.¹⁸ Studies are needed to address whether CHL1 and neurofascin play analogous roles in cancer onset and progression.The molecular mechanisms of L1-mediated signaling that govern the migration of neuronal precursors and guidance of axons during the development of the nervous system may also be used by cancer cells to facilitate invasion and cancer progression. Integrins are well-characterized cooperative partners for L1-CAMs, and signal transduction pathways activated by this complex are known to promote cell adhesion and directional motility. L1/integrin-mediated signaling may converge with growth factor signaling networks to promote motility. Like L1, CHL1 cooperates with integrins to stimulate migration. All L1-CAMs reversibly engage the actin cytoskeleton through a conserved motif FigQ/AY in the cytoplasmic domain that contains a crucial tyrosine residue required for binding the spectrin adaptor ankyrin. Phosphorylation of the FigQY tyrosine decreases ankyrin binding, whereas dephosphorylation promotes L1-ankyrin interaction. Dynamic adhesive interactions controlled by phosphorylation/dephosphorylation of the ankyrin motif in L1 family members may enable a cell to cyclically attach and detach from the ECM substrate or from neighboring cells, thus facilitating migration.¹ Another way L1 promotes cell migration is by stimulating endocytosis of integrins, reducing cell adhesion to the extracellular matrix.¹⁹ Thus, it is reasonable to speculate that upregulation of L1 in cancer may result in increased L1-mediated signaling and, consequently, increased cell migration.L1-CAMs are cleaved by metalloproteases, releasing functionally active ectodomain fragments that are laid down as “tracks” on the extracellular matrix (ECM). These fragments can cause autocrine activation of signal transduction pathways, promoting cell migration through heterophilic binding to integrins.²⁰ Specifically, L1 is cleaved constitutively or inducibly by the ADAM family metalloproteases (a disintegrin and metalloprotease) ADAM10 and ADAM17, which stimulates cell migration and neurite outgrowth during brain development.^20,21 In colon cancer, L1 colocalizes with ADAM 10 at the invasive front of the tumor tissue, suggesting that L1 shedding may play a role in cancer invasiveness.¹² Similarly, CHL1 is shed by ADAM8, which was reported to promote cell migration and invasive activity of glioma cells in vitro and is highly expressed in human brain tumors including glioblastoma multiforme, correlating with invasiveness in vivo.²² Furthermore, NrCAM, found in pancreatic, renal and colon cancers, is subject to ectodomain shedding,²³ but its function in regulating cell migration or invasion has not yet been studied.Given the newly recognized roles of L1 in tumor progression, a growing body of experimental studies has explored novel therapeutic approaches targeting L1-CAMs. Antibody-based therapeutic strategies are being pursued to functionally inhibit homophilic and heterophilic interactions of cell adhesion molecules to suppress tumor invasive motility. L1 monoclonal antibodies reduce in vivo growth of human ovarian and colon carcinoma cells in mouse xenograft models.^13,24,25 L1 targeting using lentiviral-mediated short hairpin RNA (shRNA) interference decreases growth and survival of glioma stem cells in vitro, suppresses tumor growth, and increases survival of tumor-bearing animals.¹⁴ These findings raise the possibility that L1 represents a cancer stem cell-specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors. Cancer stem cells represent a potential target for future treatment of different cancer as these cells are believed to be responsible for cancer recurrence.²⁶ Promoting cancer stem cell differentiation by drug treatment could potentially reduce stem cells properties of self-renewal and proliferation, leading to inhibition of tumor growth.Inhibitors of metalloproteases that block L1-CAM shedding represent a potentially novel approach to curtailing tumor invasiveness. Chemical inhibitors of ADAMS are appealing for glioma therapy due to their diffusability, which circumvents blood-brain barrier limitations. Another novel approach involves the secreted axon repellent protein, Semaphorin 3A (Sema3A). L1-CAMs serve as co-receptors for Sema3A by cis binding in the plasma membrane to Neuropilin-1, important for repellent axon guidance.² Interestingly, Sema3A inhibits invasiveness of prostate cancer cells²⁷ and migration and spreading of breast cancer cells in in vitro assays,²⁸ and thus may also be mediated by L1-CAMs. Such an approach could be potentially useful in mitigating invasion of cancer cells in gliomas and other tumors that are known to express L1 and Neuropilins. However, effective strategies for some types of cancer can promote cancer progression in other types. For example, Sema3A has been shown to contribute to the progression of pancreatic cancer²⁹ and colon cancer.³⁰ Thus, it is imperative that the molecular mechanisms underlying L1-mediated signaling are understood in a tissue specific manner. Despite the promising advances in cancer basic research, much more research is needed to better design strategies for cancer therapy.     

What have we learned about the kallikrein-kinin and renin-angiotensin systems in neurological disorders?

The kallikrein-kinin system(KKS) is an intricate endogenous pathway involved in several physiological and pathological cascades in the brain. Due to the pathological effects of kinins in blood vessels and tissues, their formation and degradation are tightly controlled. Their components have been related to several central nervous system diseases such as stroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis, epilepsy and others. Bradykinin and its receptors(B1R and B2R) may have a role in the pathophysiology of certain central nervous system diseases. It has been suggested that kinin B1R is up-regulated in pathological conditions and has a neurodegenerative pattern, while kinin B2R is constitutive and can act as a neuroprotective factor in many neurological conditions. The renin angiotensin system(RAS) is an important blood pressure regulator and controls both sodium and water intake. AngⅡ is a potent vasoconstrictor molecule and angiotensin converting enzyme is the major enzyme responsible for its release. AngⅡ acts mainly on the AT1 receptor, with involvement in several systemic and neurological disorders. Brain RAS has been associated with physiological pathways, but is also associated with brain disorders. This review describes topics relating to the involvement of both systems in several forms of brain dysfunction and indicates components of the KKS and RAS that have been used as targets in several pharmacological approaches.     

A chitin-like component in Aedes aegypti eggshells, eggs and ovaries

An insoluble white substance was prepared from extracts of eggshells of Aedes aegypti, the yellow fever mosquito and dengue vector. Its infrared and proton NMR spectra were similar to that of standard commercial chitin. This putative chitin-like material, also obtained from ovaries, newly laid and dark eggs, was hydrolyzed in acid and a major product was identified by HPLC to be glucosamine. The eggshell acid hydrolysate was also analyzed by ESI-MS and an ion identical to a glucosamine monoprotonated species was detected. The presence of chitin was also analyzed during different developmental stages of the ovary using a fluorescent microscopy technique and probes specific for chitin. The results showed that a chitin-like material accumulates in oocytes during oogenesis. Streptomyces griseus chitinase pre-treatment of oocytes greatly reduced the chitin-derived fluorescence. Chitinase activity was detected in newborn larvae and eggs prior to hatching. Feeding experiments indicated that the chitin synthesis inhibitor lufenuron inhibited chitin synthesis, either when mosquitoes were allowed to feed directly on lufenuron-treated chickens or when an artificial feeding system was used. Lufenuron inhibited egg hatch, larval development and reduced mosquito viability. These data demonstrate for the first time that (1) a chitin-like material is present in A. aegypti eggs, ovaries and eggshells; (2) a chitin synthesis inhibitor can be used to inhibit mosquito oogenesis; and (3) chitin synthesis inhibitors have potential for controlling mosquito populations.     

Rosuvastatin protects isolated hearts against ischemia-reperfusion injury: role of Akt-GSK-3β, metabolic environment,and mitochondrial permeability transition pore

Journal of Physiology and Biochemistry - The cardioprotective activity of rosuvastatin (R) is yet to be known. The objective of this study was to research whether R perfusion before global ischemia...     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.