Scorpion toxins: tools for studying K+ channels

Over the last period of time, a large number of scorpion toxins have been characterized. These peptidyl inhibitors of K+ channels have been very useful as probes for determining the molecular architecture of these channels, for purifying channels from native tissue and determining their subunit composition, for developing the pharmacology of K+ channels, and for determining the physiologic role that K+ channels play in target tissues. The large knowledge that we have developed regarding K+ channel function would not have been possible without the discovery of these peptidyl inhibitors. It is expected that as more novel peptides are discovered, our understanding of K+ channel structure and function will be further enhanced.     

Organization of direct hippocampal efferent projections to the cerebral cortex of the rhesus monkey: projections from CA1, prosubiculum, and subiculum to the temporal lobe

This study investigates direct hippocampal efferent projections to the temporal lobe of the rhesus monkey. Tritiated amino acid injections were placed into the hippocampal formation to identify terminal fields, and complementary fluorescent retrograde tracer injections were placed into the cortex to identify the cells of origin. Tritiated amino acid injections into CA1, prosubicular, or subicular subfields produced anterograde label over parts of the parahippocampal gyrus and temporal pole. Injections of fluorescent retrograde tracers demonstrated that these projections originate from longitudinal strips of neurons that occupy part of the CA1 subfield as well as from strips of neurons in adjacent prosubicular and subicular subfields. Thus, an injection into area TH of the posterior parahippocampal gyrus labeled neurons in a longitudinal strip of proximal CA1 (i.e., near CA2) as well as a strip in the subiculum; injections into areas TF, TL, 35, or Pro labeled neurons in a longitudinal strip of distal CA1 (i.e., near the prosubiculum) as well as one in the prosubiculum; and an injection into area TFO labeled neurons in a longitudinal strip in the middle of CA1. These strips of neurons extended longitudinally throughout the entire rostrocaudal length of the hippocampus. These results demonstrate that, in the monkey, CA1 projections to cortex arise topographically from longitudinally oriented strips of neurons that occupy only a part of the transverse extent of CA1 but that cover most of the anteroposterior extent of the hippocampus. Thus, in the monkey, CA1 is not a single uniform entity and may have a unique role as a source of direct hippocampal projections to the cerebral cortex.     

Growth factor regulation of insulin-like growth factor (IGF) binding proteins (IGFBP) and preadipocyte differentiation in porcine stromal-vascular cell cultures

The influence of anti-IGF-1 and anti-transforming growth factor beta (TGF-beta) neutralizing antibodies on preadipocyte differentiation and secretion of IGFBPs was examined in serum free porcine stromal-vascular cultures. Cultures were stained for morphological analysis and conditioned media were collected for: TGF-beta determination by ELISA, IGF-1 by RIA, and IGFBP analysis by ligand blotting. After 6 d of treatment, anti-TGF-beta increased fat proportions by 2.7 fold compared to controls. Anti-IGF-1 decreased fat cell proportions by 14-fold. Anti-TGF-beta increased concentrations of IGF-1 5.8-fold and IGFBP-2 and IGFBP-3 by 8- and 7-fold in conditioned media whereas IGFBP-4 decreased 5-fold. Anti-IGF-1 increased concentrations of IGFBP-2 and 3 by 9- and 35-fold, respectively. TGF-beta increased concentrations of IGFBP-1, 2 and 3 by 3-fold, 18-fold and 3-fold, respectively, after 9 d in culture (6 d of treatment). There was no change in TGF-beta levels in anti-IGF-1 treated cultures compared to controls. Control antibodies and negative controls had no effect. These results provide evidence that endogenously produced IGF-1 and TGF-beta has a major influence on preadipocyte differentiation in serum free media by modulating IGFBP production/secretion.     

The immediate and late allergic response to segmental bronchopulmonary provocation in asthma

The response to antigen is an important factor in the development of airway inflammation. Segmental bronchoprovocation (SBP) with antigen and subsequent bronchoalveolar lavage (BAL) have provided valuable insight into the mechanisms of allergic inflammation. To determine the features of allergic airway response in asthma, 19 subjects with mild asthma underwent antigen SBP in a dose-dependent manner. The amount of antigen used in SBP was 0 (saline), and 1, 5, or 20% of the antigen dose required to drop the FEV1 by 20% (APD20). BAL was done at 5 min and 48 h after SBP. BAL histamine levels increased modestly 5 min after antigen SBP. At 48 h, there was a marked increase in eosinophils and IL-5 concentration even in airway segments where the release of histamine was small. Moreover, eosinophils correlated with IL-5 levels at 48 h (r = 0.63; p < 0.001), but not with BAL histamine concentrations at 5 min. GM-CSF levels did not increase after antigen SBP and did not correlate with eosinophils. These observations indicate that asthmatic subjects can develop a dose-dependent response to antigen SBP that is characterized by a modest increase in histamine immediately after antigen exposure, and marked eosinophilia, which appears proportionately greater than the histamine response and relatively greater than what is seen in allergic nonasthmatic subjects. This feature might be important to the eventual development of airway inflammation in asthma.     

HLA-DM stabilizes empty HLA-DR molecules in a chaperone-like fashion

HLA-DM (DM) is a non-classical major histocompatibility complex (MHC) class II molecule that interacts with classical MHC II molecules in acidic compartments. During this association DM is supposed to catalyze the release of invariant chain (II)-derived CLIP peptides thereby rendering the peptide binding groove accessible for antigenic peptide loading. However, in situations of peptide scarcity the fate of these DM:DR complexes is not known. We could show that DR molecules incubated at lysosomal pH in the absence of peptide rapidly undergo functional inactivation and aggregation. In the presence of DM, however, empty DR molecules were shown to be stabilised and kept receptive for peptide loading, with the degree of the stabilising effect of DM varying for different DR alleles. In addition, in lysosomal compartments a considerable fraction of DM was found to be stably associated with empty DR alpha beta dimers thereby preserving their functionality. Upon encounter with antigenic peptide the DM-associated DR molecules could be rapidly loaded, whereupon they did no longer bind to DM. Thus, DM seems to act as a dedicated class II-specific chaperone that rescues uncharged alpha beta dimers. In view of the suggested shortage of self-peptides in the loading compartment, empty class II molecules that are kept receptive for loading by the chaperone function of DM may enable the antigen processing system to respond promptly to the challenge by newly entering antigens.     

Disposition of the bromosulfophthalein-glutathione conjugate in the isolated perfused rat kidney

Renal elimination of the bromosulfophthalein-glutathione conjugate (BSP-GSH) after its i.v. administration in the rat in vivo is negligible. In our study we wanted to establish whether the high albumin-binding of BSP-GSH constitutes the major restrictive factor toward the urinary excretion of the compound. The renal disposition of BSP-GSH was studied in the isolated rat kidney during perfusions with or without albumin in the perfusate. The urinary clearance of BSP-GSH in the absence of albumin was very low (< 60 microliters/min) as compared to the inulin clearance (approximately 300 microliters/min). This indicates that albumin-binding is not the major reason for the low urinary clearance of BSP-GSH. Addition of albumin to the perfusate further decreased the urinary excretion by 60%. BSP-GSH is metabolized by the kidney into two major metabolites: the cysteinylglycine conjugate and the di-glutathione conjugate. Both metabolites appear in perfusate, which suggests that BSP-GSH undergoes tubular (re-)uptake. The di-glutathione conjugate is further metabolized to the di-cysteinylglycine conjugate. The di-glutathione conjugate and the di-cysteinylglycine conjugate are the major urinary components and the urinary elimination of BSP-GSH may depend on their formation. Inhibition of gamma-glutamyl transpeptidase activity with acivicin largely prevented the degradation to the cysteinylglycine and dicysteinylglycine conjugates of BSP. The total rate of urinary excretion, however, was only slightly lowered by acivicin. Apparently, cleavage of the gamma-glutamyl moiety is not relevant for the total urinary elimination of BSP-GSH.     

Factors affecting primary care physician participation in Medicare

OBJECTIVES: This study investigates the levels of participation and the relative association of economic and noneconomic factors on primary care physician participation in the Medicare program. METHODS: Demographic information, participation in Medicare, and attitudes toward both the Medicare program and Medicare patients were collected in a written survey mailed to half the primary care physicians in Iowa. Ordinary least squares and logistic regression analyses were conducted to determine factors associated with the percentage of Medicare patients in a practice and the acceptance of all new Medicare patients, respectively. RESULTS: Two thirds of physicians were accepting all new Medicare patients, whereas 16% were accepting no new Medicare patients. Factors associated with having a higher percentage of Medicare patients in a practice were as follows: (1) a larger proportion of Medicare recipients in the county, (2) practice as a general internal medicine physician, (3) more years in practice at the current location, (4) greater enjoyment treating elderly patients, (5) less concern about having too many Medicare patients, and (6) a stronger belief that the Medicare program respects their professional judgment. Physicians less concerned about having too many Medicare patients in their practice and physicians in counties with a higher percentage of Medicare patients were significantly more likely to accept all new Medicare patients. CONCLUSIONS: These results suggest that as Medicare reforms are discussed, careful consideration of the impact of these reforms on noneconomic issues is important to ensure adequate physician participation and access for elderly patients through the Medicare program.