Evidence for interaction between transmembrane segments in assembly of Kv1.3

Previously, we showed that the N-terminal recognition domain (T1) of Kv1.3 was not required for assembly of functional channels [Tu et al. (1996) J. Biol. Chem. 271, 18904-18911]. Moreover, specific Kv1.3 peptide fragments including regions of the central core are able to inhibit expression of current produced from a channel lacking the T1 domain, Kv1.3(T1-). To elucidate the mechanism whereby Kv1.3 peptide fragments suppress Kv1.3(T1-) current, we have studied the ability of peptide fragments containing the transmembrane segments S1, S1-S2, or S1-S2-S3 to physically associate with the Kv1.3(T1-) polypeptide subunit in vitro in microsomal membranes. Using c-myc (9E10) epitope-labeled peptide fragments and anti-myc antibody as well as antisera to the Kv1.3 C-terminus, we now demonstrate specific association of these peptide fragments with Kv1.3(T1-). Association of peptide fragments with Kv1.3(T1-) was correlated with integration of both proteins into the membrane. Furthermore, the relative strength and kinetics of this association directly correlated with the ability of fragments to suppress Kv1.3(T1-) current. The rate-limiting step in the sequential synthesis, integration, and formation of a complex was the association of integrated polypeptides within the plane of the lipid bilayer. These results strongly suggest that the physical association of transmembrane segments provides the basis for suppression of K+ channel function by K+ channel peptide fragments in vivo. Moreover, the S1-S2-S3 peptide fragment potently suppressed full-length Kv1.3, thus implicating a role for the S1-S2-S3 region of Kv1.3 in the assembly of the Kv1.3 channel. We refer to these putative association sites as IMA (intramembrane association) sites.     

Molecular mechanism of base excision repair of uracil-containing DNA in yeast cell-free extracts

Base excision repair (BER) constitutes a ubiquitous excision repair mechanism, which is responsible for the removal of multiple types of damaged and inappropriate bases in DNA. We have employed a yeast cell-free system to examine the biochemical mechanism of the BER pathway in lower eukaryotes. Using uracil-containing DNA as a model substrate, we demonstrate that yeast BER requires Apn1 protein, an Escherichia coli endonuclease IV homolog. In extracts of an apn1 deletion mutant, the 5'-incision at AP (apurinic/apyrimidinic) sites is not detectable, supporting the notion that yeast contains only one major 5'-AP endonuclease. The processing of the 5'-deoxyribose phosphate moieties was found to be a rate-limiting step. During BER of uracil-containing DNA, repair patch sizes of 1-5 nucleotides were detected, with single nucleotide repair patches predominant.     

Mesopore development in PAN-ACF resulting from non-metal additives

The narrow pore-size distribution of activated carbon fibres (ACF) limits their application in the fields concerning larger molecules, such as liquid adsorption and catalyst support. On the addition of carbon particles and organic materials to polyacrylonitrile fibres, and after stabilizing and activating, the mesoporosity in the resultant ACF has been obviously increased. Among these additives, carbon black gives the best effect. With 1 wt% carbon black I in precursor and on activating at 880°C for 30 min, a mesoporosity of 48.2% (total pore volume 0.704 ml g-1, mesopore volume 0.340 ml g-1) has been obtained. The mechanism of mesopore formation is also discussed. This revised version was published online in November 2006 with corrections to the Cover Date.     

The cholinergic system in Alzheimer's disease

The past decade has witnessed an enormous increase in our knowledge of the variety and complexity of neuropathological and neurochemical changes in Alzheimer's disease. Although the disease is characterized by multiple deficits of neurotransmitters in the brain, this overview emphasizes the structural and neurochemical localization of the elements of the acetylcholine system (choline acetyltransferase, acetylcholinesterase, and muscarinic and nicotinic acetylcholine receptors) in the non-demented brain and in Alzheimer's disease brain samples. The results demonstrate a great variation in the distribution of acetylcholinesterase, choline acetyltransferase, and the nicotinic and muscarinic acetylcholine receptors in the different brain areas, nuclei and subnuclei. When stratification is present in certain brain regions (olfactory bulb, cortex, hippocampus, etc.), differences can be detected as regards the laminar distribution of the elements of the acetylcholine system. Alzheimer's disease involves a substantial loss of the elements of the cholinergic system. There is evidence that the most affected areas include the cortex, the entorhinal area, the hippocampus, the ventral striatum and the basal part of the forebrain. Other brain areas are less affected. The fact that the acetylcholine system, which plays a significant role in the memory function, is seriously impaired in Alzheimer's disease has accelerated work on the development of new drugs for treatment of the disease of the 20th century.     

Comparison of alkylglycoside surfactants in enantioseparation by capillary electrophoresis

We examined the biopsy specimens of 62 patients with diabetic nephropathy to establish whether the myofibroblast (MF) has a role in progressive interstitial fibrosis and to ascertain whether a relationship existed between MF activity and severity of arteriolosclerosis. MF were identified by morphology and alpha smooth muscle actin (alpha SMA) immunostaining. Analysis of vascular injury was performed by counting the number of interstitial arterioles after staining endothelial cells with von Willebrand factor (VWF) antibody. Arteriosclerosis was quantified by using a computer-aided image analyzer to measure the arteriolar wall surface and total arteriolar surface area, and the ratio of wall to total surface area was expressed as the index of arteriosclerosis (IA). Fractional area of interstitium (IFA), alpha SMA, and collagen III (Coll III) were quantitated by point counting. Results were related to structural and functional parameters using rank correlation coefficients. There was a strong correlation between IFA and Coll III staining (r = 0.83; P < 0.001). The alpha SMA staining correlated with IFA (r = 0.56; P < 0.001) and Coll III (r = 0.47; P < 0.001), and there were significant correlations between alpha SMA and total urinary protein (r = 0.47; P < 0.001), renal function (plasma creatinine) at time of biopsy (r = 0.51; P < 0.001), and the percent change in plasma creatinine after 4 years (delta Cr) (r = 0.37; P = 0.01). The IA correlated significantly with Coll III (r = 0.29; P = 0.02), glomerular filtration rate (GFR) (r = 0.39; P = 0.008), and creatinine (r = 0.33; P = 0.01), but no correlation was observed between alpha SMA and IA (r = 0.16; P = 0.23) or IA and delta Cr (r = -0.04; P = 0.6). Strong correlations could be shown between arteriolar density, IFA (r = 0.75; P < 0.001), alpha SMA (r = -0.36; P = 0.034), and Coll III (r = -0.66; P < 0.0001). The MF appears to have a significant role in the progression of diabetic nephropathy. Ischemia secondary to arteriosclerosis may contribute to interstitial fibrosis through fibroblast modulation into MF.     

Regional chemotherapy of liver tumors

In the absence of adequate autogenous vein for tibial artery bypass in limb salvage surgery, the use of prosthetic grafts with a distal anastomotic vein cuff or patch has shown promising results. Here, we describe how the Florester Internal Vessel Occluder (Meadox UK, Bedfordshire, UK) can facilitate the construction of a distal anastomotic vein cuff.     

Enhancing effect of natural killer cell stimulatory factor (NKSF/interleukin-12) on cell-mediated cytotoxicity against tumor-derived and virus-infected cells

Natural killer cell stimulatory factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine with pleiomorphic effects on T and NK cells, including induction of lymphokine production, mitogenesis, and enhancement of spontaneous cytotoxic activity. Similarly to IL-2, NKSF/IL-12 enhances NK cell-mediated cytotoxicity within a few hours and independently from induced proliferation. This effect is independent from other induced cytokines, because it is not prevented by antibodies neutralizing interferon (IFN)-alpha, IFN-beta, IFN-gamma, IL-2 or tumor necrosis factor (TNF)-alpha and, unlike the induction of IFN-gamma production by peripheral blood lymphocytes, it does not require HLA class II-positive accessory cells. Enhanced cytotoxicity is accompanied by morphologic changes in NK cells, including a significant increase in the number of cytoplasmic granules. In addition to the previously described ability to enhance the cytotoxic activity of NK cells against tumor-derived target cells, NKSF/IL-12 is also a potent stimulator of cytotoxicity against virus-infected cells, either fibroblasts acutely infected with herpes viruses or T cell lines chronically infected with human immunodeficiency virus-1. NK cell-mediated antibody-dependent cytotoxicity or anti-CD16 antibody-redirected lysis is not significantly enhanced by NKSF/IL-12. However, the ability of resting peripheral blood T cells to mediate anti-CD3 antibody-redirected lysis is enhanced by 18-h incubation with NKSF/IL-12, indicating that this lymphokine can modulate the cytotoxic capability of both NK and T cells.     

Significance of chloride channel activation in the gamma-aminobutyric acid induced growth hormone secretion in the neonatal rat pituitary

Growth hormone (GH) secretion of the neonatal pituitary is stimulated by tau-aminobutyric acid (GABA) (1,2). Since in most cases GABA is known to act by increasing postsynaptic membrane permeability to chloride ions we tested the importance of chloride channel activation in the GH stimulatory effect of GABA in the neonatal pituitary. In the absence of chloride in the superfusion medium GABA was without effect on GH secretion of the neonatal pituitaries and its effect was attenuated by chloride channel inhibitors. The effect of growth hormone releasing hormone (GHRH) on GH secretion was attenuated in the chloride-free media, but it was not affected by simultaneous administration of chloride channel blockers. The present study indicates that GH stimulatory effect of GABA in the neonatal pituitaries might involve chloride channel activation probably resulting in secondary activation of calcium channels.     

Prognostic factors of early sequelae and fatal outcome of Japanese encephalitis

A clinical case control study to identify prognostic factors present at hospital admission associated with early sequelae and fatal outcome of acute Japanese encephalitis (JE) was carried out in Gusi county, Henan Province, central China from June to September 1991. A total of 70 patients with laboratory-confirmed acute JE were studied, of whom 3 cases died and 33 cases had neurological or psychiatric sequelae at the end of three months follow-up. The results showed that acute JE at younger age, with higher body temperature, high white cell count in CSF, and deep coma present at hospital admission were markers for unfavorable outcomes (sequelae or fatal). A history of the vaccination was not correlated with the early sequelae and fatal outcome of the disease. The paper suggests that early diagnosis and treatment and universal JE vaccination for all susceptible populations are keys for decreasing incidence of sequelae and fatal outcome of acute JE.