Biochemistry of the autolytic processes in Antarctic krill post mortem. Autoproteolysis.

1. Autoproteolysis post mortem was examined at 0 degree C by following the changes in the major classes of krill (Euphausia superba and Euphausia crystallorophias) proteins and by liberation of peptides and free amino acids, and was based on experiments conducted on board expedition vessels in the Antarctic. 2. Primarily salt-soluble proteins were broken down during the first week of incubation, whereas water-soluble and insoluble proteins were degraded to a much smaller extent. The enzymes responsible for the hydrolysis presumably originate primarily from the digestive apparatus of the krill. 3. In general, the individual amino acids were released at rates corresponding to their relative occurrence in the bulk protein of the krill. Alanine was liberated in larger amounts than would be expected from the composition of the krill protein, and was evidently formed also by reactions other than proteolysis. Glutamic acid, and certain amino acids which presumably occur with high frequency adjacent to glumatic acid residues in the krill protein, were liberated only to a limited extent, and accumulated in smaller peptides. 4. During proteolysis, arginine seemed to be converted to some degree into ornithine, and on prolonged incubation conversion of arginine and lysine into their corresponding decarboxylation products, agmatine and cadaverine, appeared to take place.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

Transport of osmoprotective compounds in hybridoma cells exposed to hyperosmotic stress

Addition of osmoprotective compounds has a positive effect on growth and monoclonal antibody production in hyperosmotic hybridoma cell cultures. In order to better understand the processes involved in the osmoprotective response, uptake of the osmoprotective compounds glycine betaine, proline, sarcosine and glycine in mouse hybridoma cell line 6H11 during exposure to hyperosmotic stress was studied. Hyperosmotic stress (510 mOsmol/kg) was introduced through the addition of NaCl (100 mM) to the growth medium, and amino acid transport activity was measured immediately after transfer of the cells to the hyperosmotic medium. The osmoprotective capability of the four osmoprotectants tested was negatively affected if methylaminosobutyric acid (MeAiB), a specific substrate for amino acid transport system A, was simultaneously included in the hyperosmotic medium in equimolar amounts with one of the osmoprotective compounds. This was due to accumulation of MeAiB in the stressed cells, giving a significant reduction in the concentration of the osmoprotective compound inside the cells. Furthermore, addition of excess meAiB gave approx. 905 reduction in the initial rate of uptake of glycine betaine, while 40–50% reduction in the initial rate of uptake of proline, glycine and sarcosine. Similarly, addition of proline, glycine or sarcosine also gave a significant reduction in the initial rate of glycine betaine uptake. These results suggest that the four osmoprotective compounds share, at least in part, a common, MeAiB inhibitable carrier for transport into osmotically stressed hybridoma cells. This carrier is probably equal to amino acid transport system A.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

The Microenvironment of Cervical Carcinoma Xenografts: Associations with Lymph Node Metastasis and Its Assessment by DCE-MRI

Poor disease-free and overall survival rates in locally advanced cervical cancer are associated with a tumor micro-environment characterized by extensive hypoxia, interstitial hypertension, and high lactate concentrations. The potential of gadolinium diethylenetriamine pentaacetic acid-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the microenvironment and microenvironment-associated aggressiveness of cervical carcinomas was investigated in this preclinical study. CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K^trans and v_e were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a marker of hypoxia. A Millar catheter was used to measure tumor interstitial fluid pressure (IFP). The concentrations of glucose, adenosine triphosphate (ATP), and lactate were measured by induced metabolic bioluminescence imaging. High incidence of lymph node metastases was associated with high hypoxic fraction and high lactate concentration in CK-160 tumors and with high IFP and high lactate concentration in TS-415 tumors. Low K^trans was associated with high hypoxic fraction, low glucose concentration, and high lactate concentration in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between v_e and microenvironmental parameters or metastatic propensity were not detected in any of the tumor lines. Taken together, this preclinical study suggests that K^trans is a potentially useful biomarker for poor outcome of treatment in advanced cervical carcinoma. The possibility that K^trans may be used to identify patients with cervical cancer who are likely to benefit from particularly aggressive treatment merits thorough clinical investigations.     

Investigating alginate production and carbon utilization in Pseudomonas fluorescens SBW25 using mass spectrometry-based metabolic profiling

Metabolic profiling of Pseudomonas fluorescens SBW25 and various mutants derived thereof was performed to explore how the bacterium adapt to changes in carbon source and upon induction of alginate synthesis. The experiments were performed at steady-state conditions in nitrogen-limited chemostats using either fructose or glycerol as carbon source. Carbon source consumption was up-regulated in the alginate producing mutant with inactivated anti-sigma factor MucA. The mucA- mutants (also non-alginate producing mucA- control strains) had a higher dry weight yield on carbon source implying a change in carbon and energy metabolism due to the inactivation of the anti-sigma factor MucA. Both LC–MS/MS and GC–MS methods were used for quantitative metabolic profiling, and major reorganization of primary metabolite pools in both an alginate producing and a carbon source dependent manner was observed. Generally, larger changes were observed among the phosphorylated glycolytic metabolites, the pentose phosphate pathway metabolites and the nucleotide pool than among amino acids and citric acid cycle compounds. The most significant observation at the metabolite level was the significantly reduced energy charge of the mucA- mutants (both alginate producing and non-producing control strains) compared to the wild type strain. This reduction was caused more by a strong increase in the AMP pool than changes in the ATP and ADP pools. The alginate-producing mucA- mutant had a slightly increased GTP pool, while the GDP and GMP pools were strongly increased compared to non-producing mucA- strains and to the wild type. Thus, whilst changes in the adenosine phosphate nucleotide pool are attributed to the mucA inactivation, adjustments in the guanosine phosphate nucleotide pool are consequences of the GTP-dependent alginate production induced by the mucA inactivation. This metabolic profiling study provides new insight into carbon and energy metabolism of the alginate producer P. fluorescens.     

Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter⁻¹ of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low K_m value for ammonium (10 mM) and a high K_m value for l-glutamate (250 mM), and the V_max value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar K_m values (1 to 1.4 mM) and V_max values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.