Characteristics of brain Kv1 channels tailored to mimic native counterparts by tandem linkage of alpha subunits: implications for K+ channelopathies

Most neuronal Kv1 channels contain Kv1.1, Kv1.2 alpha, and Kvbeta2.1 subunits, yet the influences of their stoichiometries on properties of the (alpha)(4)(beta)(4) variants remain undefined. cDNAs were engineered to contain 0, 1, 2, or 4 copies of Kv1.1 with the requisite number of Kv1.2 and co-expressed in mammalian cells with Kvbeta2.1 to achieve "native-like" hetero-oligomers. The monomeric (Kv1.1 or 1.2), dimeric (Kv1.1-1.2 or 1.2-1.2), and tetrameric (Kv1.1-(1.2)(3)) constructs produced proteins of M(r) approximately 62,000, 120,000, and 240,000, which assembled into (alpha)(4)(beta)(4) complexes. Each alpha cRNA yielded a distinct K(+) current in oocytes, with voltage dependence of activation being shifted negatively as the Kv1.1 content in tetramers was increased. Channels containing 1, 2, or 4 copies of Kv1.1 were blocked by dendrotoxin k (DTX)(k) with similarly high potencies, whereas Kv(1.2)(4) proved nonsusceptible. Accordingly, Kv1.2/beta2.1 expressed in baby hamster kidney cells failed to bind DTX(k); in contrast, oligomers containing only one Kv1.1 subunit in a tetramer exhibited high affinity, with additional copies causing modest increases. Thus, one Kv1.1 subunit largely confers high affinity for DTX(k), whereas channel electrophysiological properties are tailored by the content of Kv1.1 relative to Kv1.2. This notable advance could explain the diversity of symptoms of human episodic ataxia I, which is often accompanied by myokymia, due to mutated Kv1.1 being assembled in different combinations with wild-type and Kv1.2.     

Multigene DNA prime-boost vaccines for SHIV89.6P

We assessed four prime-boost vaccine regimens with a Gene Gun component for SHIV89.6P in Macaca nemestrina. A dosing experiment using beta-galactosidase plasmid showed that 30 or 45 shots per dose elicited higher titer antibody than smaller doses. For SHIV89.6P, we administered a six-plasmid vaccine capable of producing non-infectious virions in vivo in combination with either vaccinia recombinants or inactivated virus. DNA prime/vaccinia boost, or the reverse, elicited strong immune responses. The SHIV89.6P challenge virus was grown in M. nemestrina peripheral blood mononuclear cells and titered in vivo intrarectally. As has been observed for SHIV89.6P in M. mulatta, the infected M. nemestrina experienced rapid and severe loss of circulating CD4+ T cells. Vaccinated macaques were challenged three weeks after the last boost. DNA prime/vaccina boost or vaccina prime/DNA boost protected 11/12 animals from acute CD4+ T cell depletion and disease, while other regimens were not effective.     

Chlorella sorokiniana immobilized on the biomatrix of vegetable sponge of Luffa cylindrica: a new system to remove cadmium from contaminated aqueous medium

A new sorption system of microalgal cells immobilized on the biostructural matrix of Luffa cylindrica for sequestering cadmium is reported. Free and immobilized Chlorella sorokiniana removed cadmium from 10 mgl(-1) solution at the efficiency of 92.7% and 97.9% respectively. Maximum cadmium sorption was observed to be 39.2 mgg(-1) at equilibrium (C(eq)) of 112.8 mgl(-1) by immobilized microalgal biomass as compared to 33.5 mgg(-1) at C(eq) of 116.5 mgl(-1) by free biomass from initial concentration of 150 mgl(-1). In continuous liquid flow column, the cadmium sorption capacity of immobilized C. sorokiniana was 192 mgg(-1), which was 73.2% of the total metal passed in 51.5 l. Metal desorption with 0.1 M HCl was 100% and the desorbed immobilized system was reusable with a similar efficiency in the subsequent cycle.     

Human lens high-molecular-weight alpha-crystallin aggregates

Alpha-crystallin high-molecular-weight (HMW) aggregates can be formed in vitro by many mechanisms, but the mechanism of in vivo aggregation has not been clearly established. HMW and LMW (low-molecular-weight) alpha-crystallins were isolated from human lenses 50-60 years of age and some spectroscopic measurements were performed. Conformational differences were suggested based on data of increased bis-ANS (4,4'-dianilino-1,1'-binaphthalene-5, 5'-disulfonic acid) and ThT (thioflavin T) fluorescence as well as increased far-UV and decreased near-UV circular dichroism (CD). These results indicated that HMW alpha-crystallin was more hydrophobic than LMW alpha-crystallin, possibly resulting from partial unfolding of alpha-crystallin. On the other hand, the increased ThT fluorescence and far-UV CD intensities indicate that an increased amount of beta-sheet conformation was involved in aggregation. These data, along with little difference in chaperone-like activity between the LMW and HMW alpha-crystallins, strongly suggest that HMW alpha-crystallin aggregates resulted from partial unfolding and disassembling-reassembling of LMW alpha-crystallin caused by posttranslational modification rather than chaperone complex formation.     

The inhibitory effect of calumenin on the vitamin K-dependent gamma-carboxylation system. Characterization of the system in normal and warfarin-resistant rats

The vitamin K-dependent gamma-carboxylation system is responsible for post-translational modification of vitamin K-dependent proteins, converting them to Gla-containing proteins. The system consists of integral membrane proteins located in the endoplasmic reticulum membrane and includes the gamma-carboxylase and the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase (VKOR), which provides gamma-carboxylase with reduced vitamin K(1) cofactor. In this work, an in vitro gamma-carboxylation system was designed and used to understand how VKOR and gamma-carboxylase work together as a system and to identify factors that can regulate the activity of the system. Results are presented that demonstrate that the endoplasmic reticulum chaperone protein calumenin is associated with gamma-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in gamma-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the gamma-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate gamma-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K(1) cofactor production by VKOR in the system where VKOR is the rate-limiting step for gamma-carboxylation. These findings put calumenin in a central position concerning regulation of gamma-carboxylation of vitamin K-dependent proteins. Reduced vitamin K(1) cofactor transfer between VKOR and gamma-carboxylase is shown to be significantly impaired in the in vitro gamma-carboxylation system prepared from warfarin-resistant rats. Furthermore, the sequence of the 18-kDa subunit 1 of the VKOR enzyme complex was found to be identical in the two rat strains. This finding supports the notion that different forms of genetic warfarin resistance exist.     

Synthesis, crystal structures and, antibacterial and antiproliferative activities in vitro of palladium(II) complexes of triphenylphosphine and thioamides

Palladium(II) complexes with triphenylphosphine (PPh₃) and thioamides of the general formulae, [Pd(L)₂(PPh₃)₂]Cl₂ and [Pd(L)₂(PPh₃)₂] have been prepared and characterized by elemental analysis, IR and NMR (¹H, ¹³C and ³¹P) methods, and two of them (trans-[Pd(PPh₃)₂(Dmtu)₂]Cl₂·(H₂O)(CH₃OH)_0.5 (1) and trans-[Pd(PPh₃)₂(Mpy)₂] (2)) by X-ray crystallography; where L = thiourea (Tu), methylthiourea (Metu), N,N′-dimethylthiourea (Dmtu), tetramethylthiourea (Tmtu), 2-mercaptopyridine (Mpy), 2-mercaptopyrimidine (Mpm) and thionicotinamide (Tna). The spectral data of the complexes are consistent with the sulfur coordination of thioamides to palladium(II). The crystal structures of the complexes show that (1) has ionic character consisting of [Pd(PPh₃)₂(Dmtu)₂]⁺² cations and uncoordinated Cl⁻ ions, while (2) is a neutral complex with Mpy behaving as anionic thiolate ligand. The coordination environment around palladium in (2) is nearly regular square-planar, while in (1) the trans angles show significant distortions from 180°. The complexes were screened for antibacterial effects, brine shrimps lethality bioassay and antitumor activity. These complexes showed significant activities in most of the cases against the tested bacteria as compared to that of a standard drug. Their antitumor activity against prostate cancer cells (PC3) is comparable with doxorubicin, together with no cytotoxic effects in brine shrimps lethality bioassay study.     

Timeline: Raising the profile of genetics in primary care

Primary care practitioners recognize that genetics is relevant to their daily practice, for example, for detecting and managing the risk of multifactorial disorders and genetic reproductive risks, and, in future, for targeted drug therapy. However, they lack confidence in their ability to apply genetic approaches. In fact, genetics is already ingrained in current practice, and the development of appropriate guidelines and web-based information resources will help practitioners to make personalized genetic risk assessment a part of holistic, patient-oriented primary health care.     

Angiotensin-(1–7) Via the Mas Receptor Alleviates the Diabetes-Induced Decrease in GFAP and GAP-43 Immunoreactivity with Concomitant Reduction in the COX-2 in Hippocampal Formation: An Immunohistochemical Study

We have previously shown that chronic treatment with angiotensin-(1?C7) [Ang-(1?C7)] can prevent diabetes-induced cardiovascular dysfunction. However, effect of Ang-(1?C7) treatment on diabetes-induced alterations in the CNS is unknown. The aim of this study was to test the hypothesis that treatment with Ang-(1?C7) can produce protection against diabetes-induced CNS changes. We examined the effect of Ang-(1?C7) on the number of cyclooxygenase-2 (COX-2) immunoreactive neurons and the glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes and assessed the changes in the neuronal growth-associated protein-43 (GAP-43) of the hippocampal formation in streptozotocin-induced diabetes in rats. Animals were sacrificed 30?days after induction of diabetes and/or treatment with Ang-(1?C7). Ang-(1?C7) treatment significantly prevented diabetes-induced decrease in the number of GFAP immunoreactive astrocytes and GAP-43 positive neurons in all hippocampal regions. Co-administration of A779, a selective Ang-(1?C7) receptor antagonist, inhibited Ang-(1?C7)-mediated protective effects indicating that Ang-(1?C7) produces its effects through activation of receptor Mas. Further, Ang-(1?C7) treatment through activation of Mas significantly prevented diabetes-induced increase in the number of the COX-2 immunolabeled neurons in all sub-regions of the hippocampus examined. These results show that Ang-(1?C7) has a protective role against diabetes-induced changes in the CNS.