A putative heterotrimeric G protein inhibits the fusion of COPI-coated vesicles. Segregation of heterotrimeric G proteins from COPI-coated vesicles

Heterotrimeric G proteins have been implicated in the regulation of intracellular protein transport, but their mechanism of action remains unclear. In vivo, secretion of chromogranin B, tagged with the green fluorescent protein, was inhibited by the addition of a general activator of trimeric G proteins (AlF4-) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Golgi apparatus. In an in vitro assay that reconstitutes intra-Golgi protein transport, we find that a membrane-bound and AlF4--sensitive factor is involved in the fusion reaction. To determine whether this effect is mediated by a heterotrimeric G protein localized to COPI-coated transport vesicles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Because G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.     

Emergency versus elective/urgent left ventricular assist device implantation

BACKGROUND: The risk and outcome in patients undergoing left ventricular assist device (LVAD) implantation on an emergency basis is still unclear. METHODS: Since April 1993, 40 patients received a Novacor and 8 patients a Heartmate LVAD in our institution. Patients with emergency LVAD placement were compared with the remainder in a retrospective manner. Parameters studied included underlying heart disease, preimplantation dysfunction of kidney, liver, lung, and cerebrum, interval of mechanical support, outcome, and complications. RESULTS: Patients with emergency LVAD placement predominantly were seen with postcardiotomy heart failure (47%) or acute myocarditis (20%) (group A) whereas elective and urgent candidates for LVAD implantation mainly had dilative cardiomyopathy (67%) or ischemic heart disease (30%) (group B). The incidence of secondary organ failure was significantly higher for all organs in group A patients (p < .01). Mean support interval in patients who underwent emergency LVAD implantation was lower (74+/-79 days vs 115+/-80 days), and fewer patients could be forwarded to heart transplantation in this group (22% vs 78%, p < .01). Moreover, bleeding complications were increased in group A (66% vs 30%, p < .01), but not thromboembolism and infection. CONCLUSION: In conclusion, the overall success rate after emergency LVAD implantation was lower, with bleeding being the most frequent complication. To achieve acceptable outcomes in disastrous situations, LVADs should be placed as early as possible.     

Isolation and analysis of a gene bbe1 encoding the berberine bridge enzyme from the California poppy Eschscholzia californica

Rats experience anorexia and reduction or cessation in growth after being provided a zinc-deficient diet. While zinc deficient, intake levels may be reduced 50% or more compared to control rats. In the present report, diurnal food intake patterns of male Sprague-Dawley rats were measured during zinc deficiency. In Study 1, rats consuming a modified AIN-93 diet were tested during the dark phase using an automated food weighing system. In zinc-deficient animals (Zn-), the onset of the first meal of the dark phase was delayed compared to zinc-adequate rats (Zn+; 106+/-47 vs. 23+/-5 min; p<0.05) and the number of meals consumed during the dark phase was reduced in Zn- vs. Zn+ rats (3.9+/-0.5 vs 7.1+/-0.4; p<0.05). In Study 2, diurnal food intake patterns were tested using a three-choice macronutrient self-selection paradigm of carbohydrate-, protein-, and fat-containing diets made deficient or adequate in zinc (1 or 30 mg Zn/kg diet). Food intake was recorded in the early-, mid-, late-dark period (4 h each) and light period (12 h). Carbohydrate intake was 70% of total intake of both Zn+ and Zn- rats during the first 5 days, but decreased significantly to 50% in the Zn- group during the last 5 days. Fat intake increased significantly in the Zn- group during the last 5 days. This increase was the result of 4 of 15 Zn- rats increasing their intake of fat significantly. Results of this study indicate that zinc status alters dark phase and macronutrient selection patterns by delaying consumption of the initial meal of the dark phase, reducing the average meal number and by changing the dominant macronutrient preference of some Zn- rats.     

Engineering the S1' subsite of trypsin: design of a protease which cleaves between dibasic residues

The serine protease trypsin was converted into a site-specific protease which hydrolyzes peptides between dibasic residues. Trypsin exhibits a high S1 specificity for Arg and Lys residues. However, the S1' specificity of trypsin is very broad, with only a slight preference for hydrophobic residues in P1'. We replaced Lys60 with Glu and Asp to introduce a high specificity for basic residues into the S1' site of trypsin. Both mutations cause a dramatic increase in the S1' specificity for Arg and Lys as measured by acyl transfer reactions. In K60E, the preference for Arg increases 70-fold while the preference for P1'-Lys increases 12-fold. In contrast, the preferences for other P1' residues either decrease slightly or remain the same. Thus, K60E prefers P1'-Arg over most other P1' residues by 2 orders of magnitude. Similar results are obtained when P1' specificity is measured in peptide cleavage assays. K60D exhibits an S1' specificity profile very similar to that of K60E, although the P1'-Arg preference is reduced by a factor of 2.5. Molecular modeling studies suggest that the high S1' specificity for Arg in K60E may be due to the formation of a salt bridge between Glu60 and the P1'-Arg of the substrate.     

Unbalanced chromosomal aberrations in neuroendocrine lung tumors as detected by comparative genomic hybridization

Typical and atypical carcinoids (TC, ATC) and small (SCLC) and large cell neuroendocrine carcinomas (LCNEC) constitute the spectrum of neuroendocrine lung tumors. Chromosomal aberrations have not been studied in LCNEC and only rarely in carcinoids. Only SCLCs have been investigated frequently for chromosomal aberrations. We compared three typical and four atypical carcinoids, one atypical carcinoid/SCLC mixed type, three SCLC, and three LCNEC for chromosomal gains and losses using comparative genomic hybridization. Typical carcinoids showed either no changes or only few chromosomal gains. Atypical carcinoids appeared genetically heterogeneous: One case had no aberrations, and three cases had few aberrations; two of them showed a deletion of 11q. SCLC and LCNEC were characterized by many gains and losses, especially similar changes of 3p, 5q, 5p, and 13q. Although ATC resemble LCNEC morphologically, there were no similarities at the genetic level. We have found a reciprocal relationship of prognosis and the amount of aberrations. TCs and ATCs with few chromosomal changes have the best prognosis, whereas SCLCs and LCNECs were generally characterized by a great amount of aberrations and worst prognosis. There was no unbalanced aberration common in all types of neuroendocrine tumors of the lung.     

Factors influencing the functional outcome of patients with acute epidural hematomas: analysis of 200 patients undergoing surgery

An actin-depolymerizing marine natural product, mycalolide B, and a related compound, kabiramide D, were labeled with biocytin, a biotin derivative, and used to specify target molecules in cultured rat 3Y1 fibroblasts. Mycalolide B exhibited the ability to bind to various intracellular proteins, probably through the Michael addition of a sulfhydryl group to C5 of mycalolide B. However, no intracellular proteins other than actin apparently reacted with biocytinylated kabiramide D, demonstrating that the binding of kabiramide D to actin was highly specific. Cells treated with biocytinylated kabiramide D followed by staining with fluorescein isothiocyanate-conjugated avidin showed that biocytinylated kabiramide D bound to stress fibers composed of F-actin, although the staining intensity was weaker than the fluorescent phalloidin staining. The assay for the binding of kabiramide D to actin, which had previously been treated with other actin-depolymerizing agents, showed that the actin-binding site for kabiramide D was the same as that for bistheonellide A, but not those for latrunculin A and cytochalasin D.     

Effects of early and late antihypertensive treatment on extracellular matrix proteins and mononuclear cells in uninephrectomized SHR

The efficacy of an early and late treatment with the angiotensin converting enzyme inhibitor lisinopril or the angiotensin II receptor blocker ICI D8731 was investigated in uninephrectomized spontaneously hypertensive rats (SHR). Rats that underwent uninephrectomy (UNX) at six weeks of age were randomly assigned to receive no treatment, lisinopril shortly after UNX, lisinopril starting 16 weeks after UNX, ICI D8731 shortly after UNX, and ICI D8731 starting 16 weeks after UNX. Blood pressure was normalized with both treatments. After six months inulin clearance was not significant different, while proteinuria and prevalence of interstitial fibrosis were significantly reduced in all treatment groups. Immunohistochemical studies revealed an interstitial, periglomerular and perivascular increase of extracellular matrix proteins in all rats, but a markedly reduced expression of collagen I, IV and fibronectin after early and late treatment compared to untreated controls. We found a significant reduction of infiltrating macrophages and T-lymphocytes in all treated animals compared to untreated controls after 2, 4 and 6 months. Especially early treatment was associated with lower numbers of infiltrating cells. Both treatments reduced proliferation of tubular and interstitial cells. There were no striking differences with regard to nephroprotection between the ACE inhibitor and angiotensin II receptor blocker. These findings show that both treatments have beneficial effects on kidney structure and function. They suggest that both ACE inhibition and angiotensin II blockade decrease renal cell proliferation and suppress the infiltration of mononuclear cells that may trigger expression of extracellular matrix proteins and progressive nephrosclerosis.     

Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy

Green fluorescent protein fused to human chromogranin B or neuropeptide Y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense-core secretory granules and may be used for time-resolved microscopy of single granules.     

Cloning and expression of the cDNA for rat NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

Following interleukin-1 (IL1) stimulation, an IL1 receptor associated kinase (IRAK) is rapidly recruited to the receptor complex. However, it is not understood if IRAK is able to interact directly with the intracellular portion of the IL1-RI or if its recruitment is mediated by a different molecule. Using the yeast two-hybrid system, we have analysed possible protein-protein interactions between IRAK, IL1-RI and IL1-RAcP. We found that IRAK is able to interact with the equivalent cytoplasmic region of the IL1-RAcP but is unable to interact with the cytoplasmic region of the IL1-RI. Immunoprecipitation of the IL1-RAcP followed by Western blot analysis using anti-IRAK antibodies revealed that IRAK co-precipitated with the IL1-RAcP. We propose that, in non-stimulated cells, IRAK is bound to the IL1-RAcP and therefore, following IL1 stimulation, both molecules are recruited simultaneously to the ILI-RI complex.