Fructose 2,6-bisphosphate levels are elevated in livers of genetically obese mice

Fructose 2,6-bisphosphate levels in freeze-clamped livers of $\text{C57BL}\text{6J}$$\text{ob}\text{ob}$ mice were 6-fold higher than the level in their lean (+/?) littermates. Overnight starvation reduced the hepatic level of this unique sugar diphosphate to 0.2 nmol/g in both the obese and lean mice. The elevated level in the obese mouse is consistent with the hyperinsulinemia of these animals.     

The role of phosphorylation in the α-adrenergic-mediated inhibition of rat hepatic pyruvate kinase

Phenylephrine in the presence of 1-methyl-3-isobutylxanthine and propanolol caused a 40–50% inhibition of pyruvate kinase (type L) activity in isolated hepatocytes, which was accompanied by a 2–3-fold increase in the phosphate content of the enzyme. These changes were blocked by the α-adrenergic antagonist dihydroergocryptine and could not be accounted for by the slight increase in cyclic AMP-dependent protein kinase activity generated by the α-adrenergic agonist. It is concluded that a significant component of the inhibition of hepatic pyruvate kinase mediated by α-adrenergic agonists can be attributed to a cyclic AMP-independent alteration in the phosphorylation state of the enzyme.     

Structure of the lysosomal sphingolipid activator protein 1 by homology with influenza virus neuraminidase

The sphingolipid activator protein 1 (SAP-1) increases the rate of hydrolysis of sphingolipids in the lysosome by apparently bringing together the substrate and the corresponding hydrolytic enzyme. This implies specific recognition of both the substrate and enzyme by SAP-1. However, binding domains in SAP-1 and recognition mechanisms involved are unknown. Amino acid sequence comparison of SAP-1 with influenza virus neuraminidase (EC 3.2.1.18, FLU NA) indicates that functional amino acid residues in or near the sialic acid binding site of FLU NA are also found at equivalent positions in the first 48 N-terminal amino acids of SAP-1. This region of homology allows to propose folding of the SAP-1 polypeptide chain by comparison with known crystallographic structure of FLU NA and identify a potential domain for lysosomal enzyme recognition through sialic acid binding. There is also a region of 10 amino acid residues near the C-terminal end of SAP-1 which has a strong propensity to form an alpha-helix with amphiphilic properties of lipid-binding helices. This domain in SAP-1 is probably responsible for the lipid(substrate)-binding function of SAP-1.     

Investigation of protein refolding: a special feature of native structure responsible for refolding ability

A possible origin of the refolding ability of globular proteins is discussed. It is shown that the structure of native proteins has a special feature, namely, that this is the only structure in which the short overlapping segments of the polypeptide chain are in one of the significantly stable conformations of the oligopeptides with the same amino acid sequences as segments themselves. It is shown that this special feature is responsible for the refolding ability of proteins. A simple formula is given for the estimation of the time, t, necessary for the spontaneous formation of a refolding nucleus by a certain segment and it is shown that the segment which has the smallest t value, will serve as a refolding nucleus. It is suggested that natural selection which ensures the maintenance of the native structure of globular proteins automatically results in the refolding ability of proteins regardless of the biological relevance of this ability.     

Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

Substrate analogue induced changes of the CO-stretching mode in the cytochrome P450cam-carbon monoxide complex.

The CO-stretching mode of the carbon monoxide ligand in reduced cytochrome P450cam, in the absence or presence of camphor and in the presence of nine different camphor analogues, was measured at room temperature using Fourier transform infrared spectroscopy. Substrate-free cytochrome P450cam--CO reveals a broad, slightly structured band resulting from an overlap of several stretching mode signals. The multitude of the signals indicates that cytochrome P450 exists in a dynamic equilibrium of several conformational substates. Binding of camphor or camphor analogues strongly influences this equilibrium. For substrate analogues which are not able to form a hydrogen bond to the hydroxyl group of tyrosine 96, the CO-stretching band is rather broad and asymmetric. In contrast, substrate analogues with one quinone group which form a hydrogen bond to the Tyr96 OH induce a shift and a sharpening of the CO-stretching mode band. For substrate analogues with two hetero groups, the infrared spectrum is slightly asymmetric or a minor band appears. Sterical hindrance, substrate mobility, and protein flexibility finally determine the position and width of the CO-stretching mode signals.     

Economic incentives for conservation: beekeeping and Saturniidae caterpillar utilization by rural communities

The economic viability of the wildlife based enterprises (bee-keeping and caterpillar utilization) in Malawi is discussed in relation to conventional agricultural enterprises (maize, beans and ground-nuts). A strong incentive emerges for rural people to adopt wildlife management as an adjunct to subsistence agriculture, and therefore, to promote conservation of natural ecosystems and wildlife habitats in the face of growing human population and demand for land. Dependence on agriculture has depleted the wildlife resource outside protected areas and has been less effective in improving the wealth and living standards of most rural people. This study illustrates that the Malawi Department of National Parks and Wildlife needs to introduce economic incentives that integrate biological conservation with economic development for the rural people. The management programme involves the adoption of a rotation burning policy that promotes vegetation coppicing, eases harvesting and promotes high caterpillar yields.     

Microcalorimetric assay of acetylcholinesterase

Comparative assays were made in a spectrophotometer and a microcalorimeter for the reaction between acetylcholinesterase (EC 3.1.1.7) and acetylthiocholine. The rate of light absorbance change and the rate of heat flow were measured from similar and simultaneous reactions in spectrophotometer and microcalorimeter, respectively. At the enzyme activity levels studied, i.e., 0.05–0.15 I.U. in calorimetry and 1–4 I.U. in spectrophotometry, the reaction rates were linear and showed first-order kinetics. A highly significant positive correlation was seen between the two methods (r = 0.997). More importantly, spectrophotometric assay with acetylthiocholine (which utilized a secondary reaction with chromagen, dithiobisnitrobenzoic acid) stood in highly significant positive correlation with calorimetric assays (which did not require a chromagen) either with the same substrate (r = 0.976) or with acetylcholine (r = 0.900). It appears that microcalorimetry can be used in preference to spectrophotometry for enzyme kinetic studies to overcome the complexity of reaction mixture and interference problems and with the advantage of using natural substrates.     

A BIOCHEMICAL-BEHAVIORAL MODEL FOR STUDYING SEROTONERGIC SUPERSENSITIVITY IN BRAIN

Chronic treatment with p-chlorophenylalanine methylester (PCPA) resulted in enhanced sensitivity to D,L-5-hydroxytryptophan (5-HTP) induced inhibition of response rates in rats working on a Variable Interval 1 min schedule of milk reinforcement. Marked depletions of 5-hydroxytryptamine (5-HT) were found in the cerebral cortex, hippocampus, striatum, diencephalon, mesencephalon, and pons-medulla oblongata. Much smaller dopamine depletions were seen in some areas, particularly the hippocampus and pons-medulla oblongata. Analysis of the binding of [³H]5-HT to crude membrane fractions from the cerebral cortex of PCPA-treated animals indicated a significant decrease in the apparent dissociation constant (K_dAPP) for 5-HT, but no change in the maximum binding capacity. The increased behavioral sensitivity to 5–HTP does not seem to be due to increased conversion of 5-HTP to 5-HT, increased uptake of 5-HTP, or release of 5-HT by p-chlorophenylethylamine. However, the possibility that p-chlorophenylalanine or the metabolite p-chlorophenylacetic acid increased the binding of 5-HT, thus decreasing K_dAPP, cannot be ruled out. Some parallels between a recently proposed model of human depression and the observations of the present investigation are discussed.