Two technetium-99m-labeled cholecystokinin-8 (CCK8) peptides for scintigraphic imaging of CCK receptors

A broad spectrum of radiolabeled peptides with high affinity for receptors expressed on tumor cells is currently under preclinical and clinical investigation for scintigraphic imaging and radionuclide therapy. The present paper evaluates two (99m)Tc-labeled forms of the C-terminal octapeptide of cholecystokinin (CCK8): sulfated (s)CCK8, with high affinity for CCK1 and CCK2 receptors, and nonsulfated (ns)CCK8, with high affinity for CCK2 receptors but low affinity for CCK1 receptors. Peptides were conjugated with the bifunctional chelator N-hydroxysuccinimidyl hydrazino niconitate (s-HYNIC). (99m)Tc-labeling, performed in the presence of nicotinic acid and tricine, was highly efficient (approximately 95%) and yielded products with a high specific activity (approximately 700 Ci/mmol) and good stability (approximately 5% release of radiolabel during 16 h incubation in phosphate buffered saline at 37 degrees C). Chinese hamster ovary cells stably expressing the CCK1 receptor (CHO-CCK1 cells) internalized approximately 3% of added (99m)Tc-sCCK8 per confluent well during 2 h at 37 degrees C. Internalization was effectively blocked by excess unlabeled sCCK8. CHO-CCK1 cells did not internalize (99m)Tc-nsCCK8. Displacement of (99m)Tc-sCCK8 and -nsCCK8 by unlabeled CCK-8 (performed at 0 degrees C to prevent internalization) revealed 50% inhibitory concentrations (IC(50)) of 8 nM and >1 microM, respectively. CHO-CCK2 cells internalized approximately 25% and approximately 5% of added (99m)Tc-sCCK8 and -nsCCK8, respectively. In both cases internalization was blocked by excess unlabeled peptide. IC(50) values for the displacement of (99m)Tc-sCCK8 and -nsCCK8 were 3 nM and 10 nM, respectively. CHO-CCK1 cell-derived tumors present in one flank of athymic mice accumulated 2.0% of injected (99m)Tc-sCCK8 per gram tissue at 1 h postinjection. This value decreased to 0.6% following coinjection with excess unlabeled peptide. Uptake of (99m)Tc-nsCCK8 was low (0.2%) and not did change by excess unlabeled peptide (0.3%). Accumulation of (99m)Tc-sCCK8 and -nsCCK8 by CHO-CCK2 cell-derived tumors (present in the other flank) amounted to 4.2% and 0.6%, respectively. In both cases uptake was significantly reduced by excess unlabeled peptide to 1.0% and 0.4% for sCCK8 and nsCCK8, respectively. Accumulation of (99m)Tc-sCCK8 was also high in pancreas (11.7%), stomach (2.0%), and kidney (2.1%), whereas uptake of (99m)Tc-nsCCK8 was high in stomach (0.7%) and kidney (1.4%). Both radiolabeled peptides showed a rapid blood clearance. In conclusion, these data show that CCK8 analogues can be efficiently labeled with (99m)Tc using s-HYNIC as chelator and nicotinic acid/tricine as coligand system without compromising receptor binding. Furthermore, the present study demonstrates that CCK1 tumors hardly accumulate (99m)Tc-nsCCK8, CCK2 tumors accumulate 2 times more (99m)Tc-sCCK8 than CCK1 tumors, and CCK2 tumors accumulate 15 times more (99m)Tc-sCCK8 than (99m)Tc-nsCCK8. Although accumulation in some nontarget organs was also higher with (99m)Tc-sCCK8, this may not reflect the human situation due to a different receptor expression pattern in humans as compared to mice. Therefore, further studies are warranted to investigate the possible use of (99m)Tc-sCCK8 for scintigraphic imaging of CCK receptor-positive tumors in humans.     

Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.

To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.     

Linking the investigations of character evolution and species diversification

Variation in diversification rates is often studied by investigating traits related to species' ecology and life history. Often, however, it is unknown whether these traits evolve gradually or in punctuated bursts during speciation. Using phylogenetic data and species' present-day trait information, we present a novel approach to assessing the mode of character change while accounting for trait-dependent speciation and extinction. Our model, "Binary-State Speciation and Extinction-node enhanced state shift" (BiSSE-ness), estimates both the rate of change occurring along lineages and the probability of change occurring during speciation, as well as independent speciation and extinction rates for each character state. Using simulations, we found that BiSSE-ness is able to distinguish along-lineage and speciational change and accurately estimate the parameters associated with character change and diversification rates. We applied BiSSE-ness to an empirical primate data set and found evidence for along-lineage changes in primate mating systems and social behaviors, whereas shifts in habitat were associated with speciation. In cases where trait changes may be linked to the speciation process itself (e.g., niche-related traits), BiSSE-ness provides a suitable framework with which to simultaneously address questions regarding species diversification and character change.     

Sinorhizobium meliloti acpXL mutant lacks the C28 hydroxylated fatty acid moiety of lipid A and does not express a slow migrating form of lipopolysaccharide

Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. Lipid A of all Rhizobiaceae is acylated with a long fatty acid chain, 27-hydroxyoctacosanoic acid. Biosynthesis of this long acyl substitution requires a special acyl carrier protein, AcpXL, which serves as a donor of C28 (omega-1)-hydroxylated fatty acid for acylation of rhizobial lipid A (Brozek, K.A., Carlson, R.W., and Raetz, C. R. (1996) J. Biol. Chem. 271, 32126-32136). To determine the biological function of the C28 acylation of lipid A, we constructed an acpXL mutant of Sinorhizobium meliloti strain 1021. Gas-liquid chromatography and mass spectrometry analysis of the fatty acid composition showed that the acpXL mutation indeed blocked C28 acylation of lipid A. SDS-PAGE analysis of acpXL mutant LPS revealed only a fast migrating band, rough LPS, whereas the parental strain 1021 manifested both rough and smooth LPS. Regardless of this, the LPS of parental and mutant strains had a similar sugar composition and exposed the same antigenic epitopes, implying that different electrophoretic profiles might account for different aggregation properties of LPS molecules with and without a long acyl chain. The acpXL mutant of strain 1021 displayed sensitivity to deoxycholate, delayed nodulation of Medicago sativa, and a reduced competitive ability. However, nodules elicited by this mutant on roots of M. sativa and Medicago truncatula had a normal morphology and fixed nitrogen. Thus, the C28 fatty acid moiety of lipid A is not crucial, but it is beneficial for establishing an effective symbiosis with host plants. acpXL lies upstream from a cluster of five genes, including msbB (lpxXL), which might be also involved in biosynthesis and transfer of the C28 fatty acid to the lipid A precursor.     

Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion

The aim of this study was to identify neurons in the subesophageal ganglion of the medicinal leech which initiate swimming activity and to determine their output connections. We found two bilaterally symmetrical pairs of interneurons, Tr1 and Tr2, located in the first division of the subesophageal ganglion which initiate swimming activity in the isolated nervous system when depolarized with brief (1-3 s) current pulses. Tr1 and Tr2 are considered trigger neurons because elicited swimming episodes outlast the stimulus duration, and because the length of elicited swim episodes is nearly independent of the intensity with which Tr1 and Tr2 are stimulated. Tr1 and Tr2 have similar morphologies. The neurites of both cells cross contralaterally in the subesophageal ganglion, project posteriorly, and exit the subesophageal ganglion in the contralateral connective. The axons of Tr1 and Tr2 extend as far posterior as segmental ganglion 18 of the ventral nerve cord. Tr1 provides direct excitatory drive to three groups of segmental neurons which are capable of initiating swimming: swim-initiating interneurons (cells 204 and 205), serotonin-containing interneurons (cells 61 and 21), and the serotonergic Retzius cells. In addition, all Retzius cells in the subesophageal ganglion are excited directly by Tr1. These three groups of neurons are excited even if Tr1 stimulation is subthreshold for swim initiation. In contrast to Tr1, Tr2 stimulation evokes transient inhibition in swim-initiating and serotonin-containing interneurons, and has little immediate effect on Retzius cells. In addition, Tr2 indirectly inhibits several oscillator neurons, including cells 208, 33, and 60. When Tr1 is stimulated during a swimming episode the swim period decreases for several cycles, while stimulation of Tr2 during swimming episodes reliably resets the ongoing swimming rhythm. Our findings indicate that Tr1 and Tr2 are trigger neurons which initiate swimming activity by different pathways. These neurons also have functional interactions with the swim oscillator network since either Tr1 or Tr2 stimulation during swimming can modulate the ongoing swimming rhythm.     

Esterase-16 (Es-16): Characterization,polymorphism, and linkage to chromosome 3 of a kidney esterase locus of the house mouse

A polymorphism for an isozyme of a presumed arylesterase, esterase-16 (EC 3.1.1.2), has been detected in kidney, heart, and spleen of the house mouse, Mus musculus, by means of isoelectric focusing and by disc electrophoresis. Three phenotypes can be distinguished: the ES-16A phenotype (IEP 5.9) was found in C57BL/10Sn and many other laboratory inbred strains; the ES-16B phenotype (IEP 6.1) was found in M. m. molossinus; and the ES-16C phenotype (IEP 5.9; very weak activity) was found in Peru-Coppock. Esterase-16 is strongly inhibited by 10^?3 m p-chloromercuribenzoate, but not by 2·10^?4 m bis-p-nitrophenyl phosphate or by 10^?3 m Diamox. It stains well with indoxyl acetate and other indigogenic substrates but only weakly with α-naphthyl acetate. Esterase-16 is completely insoluble in water. It is apparently governed by a structural gene locus, Es-16, with three alleles, Es-16 ^a , Es-16^b, and Es-16 ^c, respectively. Es-16 is closely linked to Car-1 and Car-2 on chromosome 3. Typing of 94 animals of the backcross (C57BL/10Sn × M. m. mol.) F₁ × M. m. mol. revealed a recombination frequency of 8.51±2.9%.     

Three flavonoid glycosides containing acetylated allose from stachys recta

By means of ¹³C and ¹H NMR spectroscopy three flavone glycosides, obtained from Stachys recta, were identified as 7-O-(2″-O-6″′-O-acetyl-β-D-allopyranosyl-β-D-glucopyranosides) of 4′-O-methylisoscutellarein, isoscutellarein and 3′-hydroxy-4′-O-methylisoscutellarein. The latter two compounds are isolated for the first time. Only mannose and glucose have been reported previously as sugar components of flavonoids of the genus Stachys.     

Iridoid and bisiridoid glycosides from Globularia cordifolia

From the methanolic extract of the underground parts of Globularia cordifolia, a new iridoid glycoside, 5-hydroxydavisioside (1) and a new bisiridoid glycoside, globuloside C (2) were isolated along with six known iridoid glycosides, aucubin, melampyroside, monomelittoside, globularifolin, alpinoside and asperuloside. The structures of the isolates were established by 1D and 2D NMR spectroscopy in combination with IR, UV and MS analyses.     

Regulatory and molecular properties of citrate synthases from methylotrophs

Abstract Gram-negative methylotrophs contain a high- M _r'large' citrate synthase. Gram-positive methylotrophs, on the other hand, contain a 'small' citrate synthase. These differences in M _r coincided partly with differences in NADH sensitivity. Citrate synthases from obligate Gram-negative and Gram-positive facultative methylotrophs were insensitive to feedback inhibition by NADH; only the enzymes from Gram-negative facultative methylotrophs were inhibited by NADH.