In vitro and in vivo characterization of three 68Ga- and 111 In-labeled peptides for cholecystokinin receptor imaging

Cholecystokinin (CCK) receptors are overexpressed in several human tumor types, such as medullary thyroid carcinomas and small cell lung cancers. Several ligands for the CCK2 receptor (CCK2R) have been developed for radionuclide targeting of these tumors. In this study, we evaluated whether radiolabeled DOTA-sCCK8 and its stabilized derivative, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], are suitable for imaging of CCK2R-positive tumors, using DOTA-MG0 as a reference. In vivo targeting of CCK2R-positive tumors with DOTA-sCCK8, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], and DOTA-MG0, labeled with (111)In or (68)Ga, was evaluated in BALB/c nude mice with a subcutaneous A431-CCK2R tumor. Biodistribution studies and single-photon emission computed tomography (SPECT) and positron emission tomography (PET) were performed at 1 hour postinjection. All peptides specifically accreted in the CCK2R-expressing tumors. Both (111)In-DOTA-sCCK8 and (111)In-DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)] showed good tumor retention (4.65% ID/g and 5.44% ID/g, respectively, at 4 hours postinjection). On PET/computed tomographic (CT) and SPECT/CT scans, subcutaneous A431-CCK2R tumors were clearly visualized with low uptake of sCCK8 peptides in the intestines. Whereas radiolabeled DOTA-MG0 showed high kidney uptake (70% ID/g), the sCCK8 peptides showed low uptake in the kidneys. Sulfated CCK8 analogues combined high tumor uptake with low retention in the kidney and are therefore promising tracers for imaging of CCK2R-positive tumors.     

Enzymatic enantioselective C-C-bond formation in microreactors

We have demonstrated that multiple crude enzyme lysates containing a hydroxynitrile lyase can be used for the enantioselective synthesis of cyanohydrins from aldehydes in microchannels. Using a microreactor setup, two important parameters were efficiently screened consuming only minute amounts of reagents. More importantly, results from the continuous flow reaction were fully consistent with results obtained from larger batchwise processes in which a stable emulsion was formed.     

Zn(2+) modulation of neuronal transient K(+) current: fast and selective binding to the deactivated channels

Modulation of voltage-dependent transient K(+) currents (A type K(+) or K(A) current) by Zn(2+) was studied in rat hippocampal neurons by the whole-cell patch-clamp technique. It is found that Zn(2+) selectively binds to the resting (deactivated or closed) K(A) channels with a dissociation constant (K(d)) of approximately 3 &mgr;M, whereas the affinity between Zn(2+) and the inactivated K(A) channels is 1000-fold lower. Zn(2+) therefore produces a concentration-dependent shift of the K(A) channel inactivation curve and enhances the K(A) current elicited from relatively positive holding potentials. It is also found that the kinetics of Zn(2+) action are fast enough to compete with the transition rates between different gating states of the channel. The rapid and selective binding of Zn(2+) to the closed K(A) channels keeps the channel in the closed state and explains the ion's concentration-dependent slowing effect on the activation of K(A) current. This in turn accounts for the inhibitory effect of Zn(2+) on the K(A) current elicited from hyperpolarized holding potentials. Because the molecular mechanisms underlying these gating changes are kinetic interactions between the binding-unbinding of Zn(2+) and the intrinsic gating processes of the channel, the shift of the inactivation curve and slowing of K(A) channel activation are quantitatively correlated with ambient Zn(2+) over a wide concentration range without "saturation"; i.e., The effects are already manifest in micromolar Zn(2+), yet are not saturated even in millimolar Zn(2+). Because the physiological concentration of Zn(2+) could vary over a similarly wide range according to neural activities, Zn(2+) may be a faithful physiological "fine tuner," controlling and controlled by neural activities through its effect on the K(A) current.     

Detection of infectious rotavirus in naturally contaminated source waters for drinking water production

Aims:  To assess chlorine susceptibility of Legionella pneumophila grown from two amoebic hosts, Acanthamoeba castellanii and Hartmannella vermiformis .
Methods and Results:  After being released from amoebae, Leg. pneumophila were chlorinated at 2 and 5 mg l⁻¹ for 5 min–24 h. Bacterial culturability and cytoplasmic membrane deterioration were quantified by culture assay on BCYEα agar and BacLight stains coupled with a fluorescent microscope, respectively. Chlorination reduced the culturability of Leg. pneumophila by 2·93–4·59 log CFU ml⁻¹ and damaged cellular membrane by 53·8–99·2%. Moreover, cells released from H. vermiformis exhibited significantly lower degrees in culturability reduction ( P  = 0·0008) and membrane deterioration ( P  < 0·0001) when compared with those from A. castellanii . The amoebic genus is the most significant parameter affecting cytoplasmic membrane integrity of chlorinated Legionella ( P  < 0·0001), followed by free chlorine concentration ( P  = 0·042).
Conclusions:  Legionella pneumophila replicated from H. vermiformis possess greater chlorine resistance than the cells from A. castellanii .
Significance and Impact of the Study:  This study shows the heterogeneity of amoebae-grown Leg. pneumophila in chlorine susceptibility, which should be considered in the control of legionellae proliferation, particularly in the systems where H. vermiformis is dominant, e.g. hot water plumbing.     

The La (SS-B) autoantigen, a key protein in RNA biogenesis, is dephosphorylated and cleaved early during apoptosis

In the past few years, a role for apoptotic processes in the development of autoimmune diseases has been suggested. An increasing number of cellular proteins, which are modified during apoptosis, has been described, and many of these proteins have been identified as autoantigens. We have studied the effects of apoptosis on the La protein in more detail and for the first time demonstrate that this autoantigen is rapidly dephosphorylated after the induction of apoptosis. Dephosphorylation of the La protein was observed after induction of apoptosis by several initiators and in various cell types. Furthermore, we demonstrate that at least a subset of the La protein is proteolytically cleaved in vivo, generating a 45 kDa fragment. Dephosphorylation as well as cleavage of La is inhibited by ZnSO4 as well as by several tetrapeptide caspase inhibitors, indicating that these processes require the activation of caspases. Dephosphorylation of La is inhibited by low concentrations of okadaic acid, suggesting that a PP2A-like phosphatase is involved. Generation of the 45 kDa fragment is consistent with proteolytic cleavage at amino acids 371 and/or 374. The possible significance of the apoptotic changes in the La protein for autoantibody production is discussed.     

The ‘small barbs’ Barbus humilis and B. trispilopleura of Lake Tana (Ethiopia): Are they Ecotypes of the Same Species?

Four species of small barbs (Barbus, subgenus Enteromius Cope, 1869) are known from Lake Tana, isolated in the Ethiopian highlands: B. humilis, B. trispilopleura, B. pleurogramma (all Boulenger, 1902) and B. tanapelagius de Graaf, 2000. However, only three species appear valid from cluster analysis using 32 morphometric characters and taking specimens from different locations in the southern Gulf of Lake Tana during August–October 1999. B. humilis and B. trispilopleura significantly differ from B. tanapelagius and B. pleurogramma in up to 36 characters. However, B. humilis and B. trispilopleura cannot be distinguished from each other by morphometric analysis or by gut contents. Specimens from clear, shallow rocky areas with vegetation have a darker back, will be more susceptible to birds, have significantly higher infection by cestodes, smaller size at first reproduction, lower fecundity, and correspond most to the B. trispilopleura phenotype. Specimens in turbid deeper water without vegetation are most similar to the B. humilis phenotype. We conclude that both species actually are extremes (ecotypes) of a continuum, belonging to a single biological species. The observed variation may well be induced by habitat-dependent predation pressure by birds. The high frequency (57%) of spot numbers intermediate between Boulengers number for B. trispilopleura (3) and for B. humilis (0) demonstrates the continuum best. Pigment spots and colour change in response to aquarium conditions and are in this case no valid taxonomic characters. Both characters may reduce the risk of predation. It is concluded that B. trispilopleura is a synonym of B. humilis. For future research we recommend to use the most appropriate name, B. humilis, for both types.     

Pattern of carbon dioxide production and retention is similar in adult pigs when fed hourly,but not when fed a single meal

Background

The understanding of bicarbonate kinetics and CO₂ retention in the body is necessary to conduct amino acid tracer oxidation studies in both humans and laboratory animals. Significant metabolic activity is associated with eating which can affect bicarbonate steady state kinetics. A study was conducted to assess the impact of feeding regimen on the recovery of labelled bicarbonate and energy expenditure in adult female pigs (sows). Five catheterized sows (235 ± 5 kg) were fed semi-synthetic diets as: a single meal 2 h into the infusion after an overnight fast, or in eight hourly meals starting 2 h before the infusion. Oxygen consumption, CO₂ production and ¹⁴CO₂ recovery (ie fraction not retained) were determined during primed, constant intravenous infusions of NaH¹⁴CO₃.

Results

The ¹⁴CO₂ recovery (%) after fasting (58.1 ± 4.8) was lower than that after single meal feeding (78.8 ± 5.9) or hourly meal feeding (81.0 ± 2.6, P = 0.03). CO₂ production correlated with ¹⁴CO₂ recovery during hourly feeding (r = 0.40, P = 0.01); this relationship was not significant after single meal feeding (P = 0.30), probably due to physical activity-associated CO₂ production.

Conclusions

The correlation of CO₂ retention factors with CO₂ production during hourly feeding suggests that this regimen should be preferred for future amino acid kinetics studies.