Calixarene-based phosphinic acids as inhibitors of protein tyrosine phosphatases

In the present work, the derivatives of calix[4]arene, thiacalix[4]arene, and sulfonylcalix[4]arene bearing four methylene(phenyl)phosphinic acid groups on the upper rim of the macrocycle were synthesized and studied as inhibitors of human protein tyrosine phosphatases. The inhibitory capacities of the three compounds towards PTP1B were higher than those for protein tyrosine phosphatases TC–PTP, MEG1, MEG2, and SHP2. The most potent sulfonylcalix[4]arene phosphinic acid displayed K_i value of 32?nM. The thiacalix[4]arene phosphinic acid was found to be a low micromolar inhibitor of PTP1B with selectivity over the other PTPs. The kinetic experiments showed that the inhibitors compete with the substrate for the active site of the enzyme. Molecular docking was performed to explain possible binding modes of the calixarene-based phosphinic inhibitors of PTP1B.     

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates Shaker K⁺ channels and voltage-gated Ca²⁺ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP₂ regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP₂ regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K⁺ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP₂ regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K⁺ channel family. Open-state stabilization by PIP₂ has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP₂ strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP₂ has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4–S5 linker) and R479 near the S6 activation gate are required for PIP₂ to inhibit voltage activation. The ability of PIP₂ to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP₂ on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP₂-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP₂ can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP₂ regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.     

Overexpression and one‐step renaturation‐purification of the tagged creatinine deiminase of Corynebacterium glutamicum in Escherichia coli cells

The codA gene of Corynebacterium glutamicum PCM 1945 coding for a creatinine deiminase (CDI) (EC 3.5.4.21) has been amplified and cloned. The recombinant strain of Escherichia coli that overproduces the (His)₆‐tagged inactive CDI of C. glutamicum as inclusion bodies has been constructed. After solubilization of inclusion bodies in the presence of 0.3% N‐lauroylsarcosine, the enzyme was renaturated and purified by a single‐step procedure using metal‐affinity chromatography. The yield of the (His)₆‐tagged CDI is ~30 mg from 1 L culture. The purified enzyme is sufficiently stable under the conditions designed and possesses an activity of 10–20 U/mg. The main characteristics of the tagged enzyme remained similar to that of the natural enzyme.     

Multinuclear Yeast Magnusiomyces (Dipodascus,Endomyces) magnusii is a Promising Isobutanol Producer

Higher alcohol isobutanol is a promising liquid fuel. During alcoholic fermentation, Saccharomyces cerevisiae produces only trace amounts of isobutanol. Screening the collection of nonconventional yeasts show that Magnusiomyces magnusii accumulates 440 mg of isobutanol per L in rich YPD medium. Here, the transformation protocol for M. magnusii is adapted based on the use of the dominant markers conferring resistance to nourseothricin or zeocin; the strong constitutive promoter TEF1 is cloned and a reporter system based on LAC4 gene from Kluyveromyces lactis coding for β‐galactosidase is constructed. In order to increase isobutanol production in M. magnusii, the heterologous gene ILV2 from S. cerevisiae is expressed in M. magnusii under control of the TEF1 promoter. The best stabilized transformants produce 620 mg of isobutanol per L in YPD medium and 760 mg L^?1 in the medium with 2‐oxoisovalerate. This suggests that M. magnusii is a promising organism for further development of a robust isobutanol producer.     

Alcoholic fermentation by wild-type <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> versus recombinant strains with an elevated level of intracellular glutathione

The ability of baker’s yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis, gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined. For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains.     

Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2

The prognosis of breast cancer in young women is influenced by reproductive history. Women diagnosed within 5 years postpartum have worse prognosis than nulliparous women or women diagnosed during pregnancy. Here we describe a mouse model of postpartum breast cancer that identifies mammary gland involution as a driving force of tumor progression. In this model, human breast cancer cells exposed to the involuting mammary microenvironment form large tumors that are characterized by abundant fibrillar collagen, high cyclooxygenase-2 (COX-2) expression and an invasive phenotype. In culture, tumor cells are invasive in a fibrillar collagen and COX-2-dependent manner. In the involuting mammary gland, inhibition of COX-2 reduces the collagen fibrillogenesis associated with involution, as well as tumor growth and tumor cell infiltration to the lung. These data support further research to determine whether women at high risk for postpartum breast cancer would benefit from treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) during postpartum involution.     

Nicotinamide‐rich diet improves physical endurance by up‐regulating SUR2A in the heart

SUR2A is an ATP‐binding protein that serves as a regulatory subunit of cardioprotective ATP‐sensitive K⁺ (K_ATP) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled K_ATP channels, we have suggested that nicotinamide‐rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide‐rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild‐type mice on nicotinamide‐rich diet. The experiments focused on action membrane potential and intracellular Ca²⁺ concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal K_ATP channels and steady Ca²⁺ levels in cardiomyocytes in response to β‐adrenergic stimulation. In contrast, the same challenge in the wild‐type was characterized by a lack of the channel activation and rise in intracellular Ca²⁺. Nicotinamide‐rich diet was ineffective to increase physical endurance in mice lacking K_ATP channels. This study has shown that nicotinamide‐rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide‐rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable.     

Differences in the substrate specificity of glycosyltransferases involved in landomycins A and E biosynthesis

A lanGT4 mutant of the landomycin A producer Streptomyces cyanogenus S136 was constructed, leading to the production of landomycin D with two deoxy sugars in the side chain and proving that LanGT4 is responsible for attaching the third deoxy sugar of the hexasaccharide side chain. Heterologous expression of lndGT4 of the landomycin E producer Streptomyces globisporus 1912 in the lanGT4 mutant restored landomycin A production, indicating that LndGT4, like LanGT4, also has the ability to work iteratively. A S. cyanogenus S136 mutant with a mutation in lanGT1, encoding a d-olivosyltransferase, was shown to produce landomycin I with one deoxy sugar and, surprisingly, a new landomycin derivative (landomycin L) containing a d-olivose followed by an l-rhodinose. Heterologous expression of lndGT1 of S. globisporus 1912 in the lanGT1 mutant did not restore landomycin A production but led to the formation of a second new landomycin derivative (landomycin K) containing an unusual pentasaccharide chain (d-olivose–d-olivose–l-rhodinose–d-olivose–l-rhodinose). The formation of landomycin L and landomycin K is most probably attributed to the high substrate flexibility of the rhodinosyltransferase LanGT4. A. Erb and C. Krauth contributed equally to this work.