Improved monoterpene biotransformation with <Emphasis Type="Italic">Penicillium</Emphasis> sp. by use of a closed gas loop bioreactor

A closed gas loop bioprocess was developed to improve fungal biotransformation of monoterpenes. By circulating monoterpene-saturated process gas, the evaporative loss of the volatile precursor from the medium during the biotransformation was avoided. Penicillium solitum, isolated from kiwi, turned out to be highly tolerant towards monoterpenes and to convert α-pinene to a range of products including verbenone, a valuable aroma compound. The gas loop was mandatory to reproduce the production of 35 mg L⁻¹ verbenone obtained in shake flasks and also in the bioreactor. Penicillium digitatum DSM 62840 regioselectively converted (+)-limonene to the aroma compound α-terpineol, but shake flask cultures revealed a pronounced growth inhibition when initial concentrations exceeded 1.9 mM. In the bioreactor, toxic effects on P. digitatum during biotransformation were alleviated by starting a sequential feeding of non-toxic limonene portions after a preceding growth phase. Closing the precursor-saturated gas loop during the biotransformation allowed for an additional replenishment of limonene via the gas phase. The gas loop system led to a maximum α-terpineol concentration of 1,009 mg L⁻¹ and an average productivity of 8–9 mg L⁻¹ h⁻¹ which represents a doubling of the respective values previously reported. Furthermore, a molar conversion yield of up to 63% was achieved. M. Pescheck and M. A. Mirata have contributed equally to this work.     

Functional consequences of DECTIN-1 early stop codon polymorphism Y238X in rheumatoid arthritis

Introduction

We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.

Methods

Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.

Results

Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.

Conclusions

These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.     

Structure of the DNA-bound BRCA1 C-terminal Region from Human Replication Factor C p140 and Model of the Protein-DNA Complex

BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5′-phosphorylated double-stranded DNA. The replication factor C BRCT domain possesses a large basic patch on one face, which includes residues that are structurally conserved and ligate the phosphate in phosphopeptide binding BRCT domains. An extra α-helix at the N terminus, which is required for DNA binding, inserts into the major groove and makes extensive contacts to the DNA backbone. The model of the protein-DNA complex suggests 5′-phosphate recognition by the BRCT domains of bacterial NAD⁺-dependent ligases and a nonclamp loading role for the replication factor C complex in DNA transactions.     

Identifying potential survival strategies of HIV-1 through virus-host protein interaction networks

Background  

The National Institute of Allergy and Infectious Diseases has launched the HIV-1 Human Protein Interaction Database in an effort to catalogue all published interactions between HIV-1 and human proteins. In order to systematically investigate these interactions functionally and dynamically, we have constructed an HIV-1 human protein interaction network. This network was analyzed for important proteins and processes that are specific for the HIV life-cycle. In order to expose viral strategies, network motif analysis was carried out showing reoccurring patterns in virus-host dynamics.     

Cytoplasmic retention of protein tyrosine kinase 6 promotes growth of prostate tumor cells

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is nuclear in epithelial cells of the normal prostate, but cytoplasmic in prostate tumors and in the PC3 prostate tumor cell line. The impact of altered PTK6 intracellular localization in prostate tumor cells has not been extensively explored. Knockdown of endogenous cytoplasmic PTK6 resulted in decreased PC3 cell proliferation and colony formation, suggesting that cytoplasmic PTK6 stimulates oncogenic pathways. In contrast, reintroduction of PTK6 into nuclei of PC3 cells had a negative effect on growth. Enhanced tyrosine phosphorylation of the PTK6 substrate Sam68 was detected in cells expressing nuclear-targeted PTK6. We found that mechanisms regulating nuclear localization of PTK6 are intact in PC3 cells. Transiently overexpressed PTK6 readily enters the nucleus. Ectopic expression of ALT-PTK6, a catalytically inactive splice variant of PTK6, did not affect localization of endogenous PTK6 in PC3 cells. Using leptomycin B, we confirmed that cytoplasmic localization of endogenous PTK6 is not due to Crm-1/exportin-1 mediated nuclear export. In addition, overexpression of the PTK6 nuclear substrate Sam68 is not sufficient to bring PTK6 into the nucleus. While exogenous PTK6 was readily detected in the nucleus when transiently expressed at high levels, low-level expression of inducible wild type PTK6 in stable cell lines resulted in its cytoplasmic retention. Our results suggest that retention of PTK6 in the cytoplasm of prostate cancer cells disrupts its ability to regulate nuclear substrates and leads to aberrant growth. In prostate cancer, restoring PTK6 nuclear localization may have therapeutic advantages.Key words: PTK6, BRK, Sik, SAM68, prostate cancer, nuclear localization, tyrosine kinase     

CTGF expression during mouse embryonic development

Connective tissue growth factor (CTGF) is a potent fibroblast mitogen and angiogenic factor which plays an important role in wound healing, cancerogenesis and fibrotic and vascular disease. Here we explored the regulation and the cellular site of the mRNA synthesis for this growth factor in the developing mouse embryo by in situ hybridisation. Strong and persistent CTGF gene expression was limited to three types of tissue: the vascular endothelium, particularly the high-pressure part of the cardiovascular system, condensed connective tissue around bone and cartilage, and maturing layer VII neurons in the cerebral cortex. With few exceptions (late tooth bud, neuroepithelium) epithelial tissue was negative. Very transient but strong expression was observed early during formation of cartilage, in late stages during perichondral ossification, on cerebral neuroepithelium, and in several discrete stages of tooth formation, on mesenchymal precursors of odontoblasts condensing on inner dental epithelium, and later on apposing regions of ameloblast and odontoblast epithelium. Altogether, the current study suggests that CTGF performs a dual role: a continuous function in the cardiovascular system, bone and cartilage-associated mesenchyme and maturing layer VII neurons, but also a more transient function associated with the formation of cartilage, bone, tooth and cerebral nerve cells.     

Free calcium ion concentration in the sieve-tube sap of Ricinus communis L. seedlings

Changes in free Ca²⁺ in sieve-tube sap have been proposed to be important in the regulation of phloem transport, and Ca²⁺-activated protein kinase activity has been described in phloem exudate (S.A. Avdiushko et al. 1997 J Plant Physiol 150: 552–559). Using atomic absorption spectrometry, we have determined that the total Ca²⁺ concentration in sieve-tube sap from Ricinus seedlings containing the endosperm is about 100 μM (range 80–150 μM). We used three independent methods to determine the free calcium ion concentration in the phloem sap ([Ca²⁺]_p). The first method was to calculate [Ca²⁺]_p from the total Ca²⁺ concentration, in combination with the binding constants and concentrations of the ionic solutes in phloem sap. The resultant estimate of [Ca²⁺]_p was 63 μM. The second method used the Ca-specific fluorescent dye 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic-acid (FURAPTRA) on exuded sieve-tube sap. Although the sap interfered severely with the fluorescence properties of the dye, Ca²⁺ titrations enabled a value of [Ca²⁺]_p = 20 μM to be deduced. The third method used Ca²⁺-selective microelectrodes on exuded sap samples, which gave an average value for [Ca²⁺]_p = 13 μM. No significant change in this value was observed during the sap exudation period. The Ca²⁺ buffer capacity was determined and the result of about 0.6 mmol · l⁻¹ · pCa⁻¹ displayed excellent agreement with the measured values of free and total Ca²⁺ concentration in sieve-tube sap. Since the measured values for free Ca²⁺ are 20- to 100-fold higher than those usually reported for the cytosol of a range of plant cells in resting conditions, it is concluded that either regulation of [Ca²⁺]_p is of limited physiological importance, or that the Ca²⁺-dependent proteins respond only to relatively high [Ca²⁺]_p. The implications for regulation of cytosolic free Ca²⁺ in symplastically connected companion cells is discussed. Received: 15 February 1998 / Accepted: 14 March 1998     

Effects of ATP analogs on the proton pumping by the vacuolar H+-ATPase from maize roots

Understanding the regulatory properties of the activities of the V-type adenosine triphosphatase (ATPase) on tonoplast membranes is important in determining the mechanisms by which this enzyme controls cytoplasmic and vacuolar pH. The possible existence of a regulatory site for adenine nucleotides was examined by comparing the effects of ADP, adenylylimidodiphosphate (AMP-PNP) and 3'- o -(4-benzoyl) benzoyladenine 5'-triphosphate (BzATP) to those of the 2',3'-dialdehyde derivative of AMP (oAMP) and ATP by using highly purified tonoplast vesicles from maize ( Zea mays L. cv. FRB 73) roots. The addition of either AMP-PNP or BzATP reversibly inhibited the initial rate of proton transport catalyzed by the H⁺-ATPase in a concentration-dependent manner. Less than 20 μ M AMP-PNP or 50 μ M BzATP was sufficient to inhibit half the initial rate of proton transport in the presence of 2 m M ATP and an excess of Mg. Both analogs increased the K_m for ATP and reduced the maximum enzyme velocity. The presence of ADP also inhibited proton transport. The characteristics of ADP-induced inhibition were similar to those of BzATP and AMP-PNP. The addition of the periodated derivative of AMP (oAMP) irreversibly inhibited the ATPase in a concentration and time-dependent manner similar to that reported previously (Chow et al. 1992, Plant Physiology 98: 44–52). Irreversible inhibition by oAMP reduced the maximum velocity of the tonoplast ATPase and was prevented by the addition of ATP. The presence of ADP, AMP-PNP or BzATP had no effect on irreversible inhibition by oAMP. The effects of ADP, AMP-PNP and BzATP on the kinetics of ATP utilization and the lack of protection against inhibition by oAMP argue in favor of at least two types of nucleotide binding sites on the V-type ATPase from maize root tonoplast membranes.     

Evidence for an indirect coupling mechanism for the nitrate-sensitive proton pump from corn root tonoplast membranes

The nitrate-sensitive proton-translocating adenosine triphosphatase (H⁺-ATPase) of tonoplast membranes plays an important role in regulating the flow of nutrients and metabolic waste between the cytoplasm and vacuole in the cells of plant roots. Relatively little information is available regarding the coupling between ATP hydrolysis and proton pumping by the nitrate-sensitive, tonoplast H⁺-ATPase. The coupling may be achieved either directly, i. e. the two reaction pathways share at least one common molecular step, or indirectly, i. e. the two reaction pathways do not share an intermediate step. These coupling mechanisms may be differentiated by the responses of the two events to external perturbation. The effects of the presence of nitrate in the assay medium on the rates of ATP hydrolysis and proton transport catalyzed by the tonoplast H⁺-ATPase from maize ( Zea mays L. cv. FRB 73) were investigated. The presence of nitrate inhibited proton transport activity of the tonoplast H⁺-ATPase to a much greater degree than ATP hydrolysis. This differential response of the two activities to nitrate is the basis for a proposed reaction model for the tonoplast H⁺-ATPase that features an indirect coupling mechanism between ATP hydrolysis and proton transport.