L-Ala-substituted rat galanin analogs distinguish between hypothalamic and jejunal galanin receptor subtypes

In order to explore which amino acids or which blocks of amino acids in the 29 amino acid neuropeptide galanin are important for recognition of the endogenous ligand by galanin receptor subtypes present in the jejunum and in the hypothalamus, respectively, we have carried out L-Ala substitutions of individual amino acids or of blocks of amino acids in the rat galanin sequence and examined the binding of the obtained analogs to the rat hypothalamic and jejunal galanin receptor subtypes. This study reveals that the galanin sequence YLLGPH^9–14 is essential for recognition of galanin by both the rat hypothalamic and jejunal galanin receptor subtypes. Substitution of the N-terminal amino acids, GWTL^1–4, leads to total loss of affinity of galanin for both hypothalamic and jejunal galanin receptors. The α-helical C-terminal amino acid (25–29) part of galanin has no greater influence on the affinity of galanin to the hypothalamic galanin receptor subtype. L-Ala substitution of the C-terminal amino acids of galanin KHGLT^25–29 shows, however, that this C-terminal motif is essential for the recognition by the jejunal galanin receptor subtype, whereas amino acids in the middle portion of galanin NSAG^5–8 are of importance for binding to the hypothalamic but not to the jejunal receptor. [Ala^5–8] Galanin thus has a more than 100-fold higher affinity to jejunal receptor than to the hypothalamic receptor, while [Ala^25–29] galanin has a more than 100-fold higher affinity for the hypothalamic than for jejunal galanin receptor subtypes. pH dependence of the galanin binding to these receptor subtypes is also different. © Munksgaard 1997.     

FLOW REQUIREMENTS AND REBREATHING DURING MECHANICALLY CONTROLLED VENTILATION IN A T-PIECE (MAPLESON E) SYSTEM

The influence of fresh gas flow (FGF) setting on rebreathingwas investigated in 15 infants and children (weight 3.5–21.8kg) during balanced anaesthesia with mechanically controlledventilation using a T-piece (Mapleson E) system and a Nuffieldventilator 200. Tidal volume (VT), minute volume (VE), maximalinspired (PlCO₂) and end-tidal (PE'_co2) carbon dioxide tensionsand airway pressure were measured. VE, set to produce a PE'_CO2of about 4.5 kPa and measured at a high FGF (minimal rebreathing),was unchanged throughout the study and the regression equationfor VE and weight was: VE (ml min^–1) = 146x kg ³482, r = 0.92. Measurements were then repeated at FGF: VE ratiosreduced to 1.5 and 1.0. To achieve minimal rebreathing (Pl_CO2less than 0.5 kPa), FGF: VE ratios greater than 1.8 (range 1.8–4.9)had to be used. At FGF: VE ratios of 1.5, some alveolar rebreathingoccurred, indicated by increased inspired (P < 0.001) andend-tidal (P < 0.001) carbon dioxide tensions. At FGF: VEratios equal to 1.0, alveolar rebreathing was more pronouncedand hypercapnoea occurred with a PE'_CO2 (mean ± 1 SD)of 5.89±0.53 kPa. At this FGF setting, change in I: Eratio from 1: 2 to 1: 1 did not influence the level of alveolarrebreathing. A minimal FGF (ml min^–1) setting of 1.5 xVE (that is, 1.5 (146 x kg ³ 482), approximated to theexpression (200 x kg ³ 1000) is recommended for controlledventilation to avoid hypercapnoea when using the T-piece systemin children weighing less than 20 kg.     

Acute Myocarditis

ABSTRACT In a prospective study, 57 patients with a preliminary diagnosis of myocarditis were investigated. Twenty-four patients were considered to have an acute myocarditis, 14 had a suspected myocarditis, while in 19 patients myocarditis was excluded. Episodes of frequent supraventricular and/or ventricular extrasystoles during hospital stay were seen in 8/24 cases (33%) with myocarditis and in 1/19 cases (5%) without myocarditis. On follow-up 1 month later, no supraventricular extrasystoles were observed in either group. Echocardiographic signs consistent with left ventricular insufficiency were noted in 7/24 cases (29%) with myocarditis, in 1/14 cases (7%) with suspected myocarditis and in no case without myocarditis. With a “routine” serologic test battery covering influenza viruses A and B, adenovirus, Coxsackie virus group B, ECHO viruses, Chlamydia psittaci, Mycoplasma pneumoniae and hemolytic streptococci group A, a possible etiology could be documented in 9/24 cases (38%) with myocarditis and in 4/19 cases (21%) without myocarditis. Enterovirus-specific IgM was detected with solid-phase reverse immunosorbent test (SPRIST) in 12/23 (48%) cases with myocarditis and in 3/16 cases (19%) without myocarditis. In SPRIST-IgM-positive cases, IgM antibodies were detected in 15/20 (75%) of the sera taken on admission. The overall serological results indicated a recent infection in 16/24 cases (67%) with myocarditis and in 5/19 cases (26%) without myocarditis (p < 0.05).     

Orthogonal solid-phase synthesis of a monobiotinylated analog of Neuropeptide Y

Analogs of Neuropeptide Y (NPY) were synthesized with conventional Boc/benzyl protective group strategy. Instead of Asn⁷ in the native scquence, Boc-Lys(Alloc)-OH was incorporated. At the end of the synthesis the Alloc group was selectively removed by palladium-catalyzed hydrostannolysis and biotin coupled to the e-amino group of Lys⁷. After cleavage and characterization with plasma desorption mass spectrometry the N^e-7-biotinyl-[Lys⁷]-NPY and the nonbiotinylated analog [Lys⁷]-NPY were investigated as ligands to the NPY receptor from rat cerebral cortex. Both analogs were found to be high affinity ligands to the NPY receptor and bound with essentially the same affinity as unmodified NPY.     

Site-specific biotinylation

We have developed an expeditious method for the incorporation of the biotinylaminocaproyl moiety on the ε-amino group of a lysine residue within a peptide chain in a site-specific manner. Using t-Boc chemistry for the solid phase synthesis approach and a base labile, acid stable protecting group (Fmoc-) for the ε-amino group of the target lysine, we prepared fully protected resin bound peptides which are site-specifically biotinylated. Following HF cleavage, the uniquely biotinylated peptides were obtained in a high degree of purity. Using this approach, a number of biotinylaminocaproyllysyl derivatives of a monocyclic Endothelin-1 analog were prepared. Synthesis of selected bicyclic analogs of high affinity monocycles led to the preparation of the bicyclic [Nle⁷]ET-1 analog containing ε-biotinylaminocaproyllysine at position-9. This peptide, with K_d= 0.08 nM, has 1000-fold higher affinity for the ET_A receptor than the commercially available N_α-biotinylated Endothelin-1. The general utility of this biotinylation methodology was demonstrated by the synthesis of a site-specifically biotinylated PTH analog which contained several side chain functionalized amino acid residues in its sequence. The synthetic method reported here is convergent in that it allows the facile variation of the length of the spacer and also offers the potential to introduce in a site specific manner other groups such as affinity labels and fluorescent tags.     

LONGITUDINAL COMPARISON OF CARBOHYDRATE-DEFICIENT TRANSFERRIN AND GAMMA-GLUTAMYL TRANSFERASE: COMPLEMENTARY MARKERS OF EXCESSIVE ALCOHOL CONSUMPTION

The utility of carbohydrate-deficient transferrin (CDT) andgamma-glutamyl transferase (GGT) as biochemical markers of excessivealcohol consumption was studied in alcohol-dependent subjects.Serum samples were collected once weekly from 10 male out-patientsundergoing a 6-month alcohol treatment programme. Frequencyof relapse into drinking (defined as any intake of alcoholicbeverage) was assessed by self-reports during patient interviewsthree times per week and by daily determination of the 5-hydroxytryptophollevel in urine. A marked decrease in mean CDT and GGT valueswas observed during the initial month. Only one patient remainedtotally abstinent throughout the observation period, while fourhad sporadic relapses (2–5 days with alcohol consumption).Both CDT and GGT remained below the respective reference limitsin those patients. The other five patients drank more frequently(range 22–57 days) and increased their mean levels ofCDT and GGT after the initial decrease. As determined from thevalues at admission and during the course of the study, CDTappeared to be the most sensitive marker in six out of the 10patients. In one patient, both markers were affected in a parallelway, whereas two of those with frequent relapses responded toalcohol consumption with a marked increase in GGT, but withno or only a slight increase in CDT. One patient did not showany abnormal CDT or GGT values. In 54 female and 60 male serumsamples collected at random from patients during admission atan alcohol detoxification unit, 35% and 58% of the CDT valuesexceeded the reference limits for females and males, respectively.For GGT, 59% of the female and 67% of the male values were abovecut-off. Carbohydrate-deficient transferrin and GGT were notsignificantly correlated. Taken together, the present resultsindicate that measurement of both CDT and GGT will increasethe possibility of identifying excessive alcohol consumption.By following changes in CDT and GGT values during a period ofalcohol withdrawal, the most sensitive individual marker canbe determined. This in turn allows for improved detection ofrelapse into heavy drinking dunng long-term monitoring of out-patients.     

Concentration- and Region-dependent Intestinal Permeability of Fluvastatin in the Rat

The purpose of this study was to investigate the mechanisms of transport of fluvastatin across the intestinal mucosa in various regions of the intestine in the rat. In-situ single-pass perfusions of the jejunum, ileum and colon were performed and the effective permeability (P_eff) of fluvastatin, antipyrine and D-glucose were assessed in each region, at three different perfusate fluvastatin concentrations (1.6, 16 and 160 μM). The effect of lovastatin acid on the bi-directional transport of fluvastatin across the ileal mucosa was also studied. The P_eff of fluvastatin was found to be dependent both on the intestinal region and on the concentration in the intestinal lumen (P < 0.001). Fluvastatin had the lowest P_eff (0.55±0.10 times 10^?4 cm s^?1) in the jejunum at 1.6 μM, and the highest P_eff (1.0±0.16 times 10^?4 cm s^?1) in the colon at 160 μM. The highest concentration of fluvastatin increased the average absorption of water from the intestine by 209% (P < 0.05), and the average P_eff of D-glucose by 29% (P < 0.05). The presence of excess lovastatin acid (100 μM, compared with fluvastatin 1.6 μM) at the luminal side increased the average absorption of water by 218% (P < 0.001), and the P_eff of fluvastatin in the ileum and the colon by 44 and 50%, respectively (P < 0.05). The presence of lovastatin acid on the luminal side in the ileum also increased the blood-to-lumen transport (exsorption) of fluvastatin by 43% (P < 0.001). The increased intestinal absorption of fluvastatin at higher concentrations does not suggest that substantial absorption occurs by any carrier-mediated process in the absorptive direction. The increased bi-directional transport when lovastatin acid was added to the lumen suggests that fluvastatin is not a P-glycoprotein substrate. Instead, the concentration-dependent increase in the absorption of fluvastatin, water and D-glucose suggests a direct effect of fluvastatin on the transcellular passive transport.