Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro-, 4-bromo-, and 4-iodobenzoate by Alcaligenes denitrificans NTB-1.

Alcaligenes denitrificans NTB-1, previously isolated on 4-chlorobenzoate, also utilized 4-bromo-, 4-iodo-, and 2,4-dichlorobenzoate but not 4-fluorobenzoate as a sole carbon and energy source. During growth, stoichiometric amounts of halide were released. Experiments with whole cells and cell extracts revealed that 4-bromo- and 4-iodobenzoate were metabolized like 4-chlorobenzoate, involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoate, which in turn was hydroxylated to 3,4-dihydroxybenzoate. The initial step in the metabolism of 2,4-dichlorobenzoate was catalyzed by a novel type of reaction for aerobic organisms, involving inducible reductive dechlorination to 4-chlorobenzoate. Under conditions of low and controlled oxygen concentrations, A. denitrificans NTB-1 converted all 4-halobenzoates and 2,4-dichlorobenzoate almost quantitatively to 4-hydroxybenzoate.     

Conformational states of (K+ + H+)-ATPase studied using tryptic digestion as a tool

The (K+ + H+)-ATPase from gastric mucosa has been treated by limited proteolytic digestion with trypsin to study the conformational states of the enzyme. The existence of a K+- and an ATP-form of the enzyme follows from the kinetics of inactivation and from the specific cleavage products. In the presence of K+ the 95 kDa chain is cleaved into two fragments of 56 and 42 kDa, whereas in the presence of ATP fragments of 67 and 35 kDa are formed. When Mg2+ is present during tryptic digestion cleavage products which are specific for both the ATP- and the K+-form of the enzyme are yielded. In analogy to ATP, Mg2+ is able to convert the enzyme from a K+-conformation to a more protected form. Moreover Mg2+ supports the protecting effect of ATP against tryptic inactivation. The K0.5 for ATP is lowered from 1.6 mM (no Mg2+) to 0.2 mM in the presence of 10 mM Mg2+. Mg2+, which in previous studies has been shown to induce a specific conformation, apparently induces a conformation different from the K+-form of the enzyme and has ATP-like effects on the enzyme. In addition it has been found that in the initial rapid phase of the digestion process the K+-ATPase activity is interrupted at a step which is very likely the interconversion of the phosphoenzyme forms E1P and E2P, since neither the K+-stimulated p-nitrophenylphosphatase activity nor the phosphorylation of the enzyme are inhibited in this phase. During the tryptic digestion in the presence of K+ there is a good correlation between the residual ATPase activity and the amount of the catalytic subunit left, suggesting that the latter is homogeneous. After tryptic digestion in the presence of K+, phosphorylation only occurs in the 42 kDa and not in the 56 kDa band. The same experiments in the presence of ATP yield only phosphorylation in the 67 kDa band and not in the 35 kDa band. A provisional model for the structure of the catalytic subunit is given.     

Calcium-induced changes in permeability of dioleoylphosphatidylcholine model membranes containing bovine heart cardiolipin

At calcium concentrations up to about 4 mM a selective permeability increase of cardiolipin/dioleoylphosphatidylcholine (50:50, mol%) membranes for calcium and its chelator arsenazo III is observed. Under these conditions calcium does not occupy all the binding sites of cardiolipin at the membrane interface and no vesicle-vesicle interactions are found. Lowering of the cardiolipin content of the vesicles to 20 mol% extends the calcium concentration range in which a selective permeability for calcium and arsenazo III is appearing up to about 12 mM. We suggest that the observed selective permeability increase is caused by transient formation of inverted micellar structures in the membrane with cardiolipin as translocating membrane component for calcium and arsenazo III. At calcium concentrations of 4 mM and higher for 50 mol% cardiolipin-containing vesicles a general permeability increase is found together with calcium-cardiolipin binding in a 1:1 stoichiometry, vesicles aggregation and, above 8 mM of calcium, vesicle fusion. The loss of barrier function of the membrane under these conditions is correlated with vesicle aggregation and may be explained by a transition from a bilayer into a hexagonal HII organization of the phospholipids.     

The effect of vitreous humour on prostaglandin production by cultured rabbit chorioretinal fibroblasts

Factors in vitreous humour which regulate prostaglandin production were investigated using cultured rabbit chorioretinal fibroblasts. These cells produced predominantly prostaglandin E2, 6-ketoprostaglandin F1 alpha, a compound likely to be a metabolite of prostaglandin E2 and 5-hydroxyeicosatetraenoic acid. The synthesis of 6-ketoprostaglandin F1 alpha was nearly completely inhibited by the cyclooxygenase inhibitor aspirin and partially inhibited by 10(-6) M dexamethasone (49%) and 10(-5) M forskolin (68%). Addition of 10% rabbit vitreous humour to subconfluent cells maintained in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum resulted in stimulation of 6-ketoprostaglandin F1 alpha production by as much as 246% as measured by radioimmunoassay. Chorioretinal fibroblasts labelled by [3H]arachidonic acid incorporation into cellular phospholipids synthesised greater amounts of all labelled arachidonic acid metabolites in response to vitreous humour. It was concluded, therefore, that there are factors present in vitreous humour of molecular weight above 10 kDa which are capable of stimulating cellular cyclooxygenase activity. Confluent cells also responded to a factor(s) present in vitreous humour. The fraction of less than 10 kDa inhibited 6-ketoprostaglandin F1 alpha production by 50% when used at a concentration of 10%. Furthermore, 6-ketoprostaglandin F1 alpha production in confluent cells (but not subconfluent cells) was inhibited to 40% of control levels by vitamin C at a concentration of 1 mg/100 ml. The latter result points to an inhibitory role for vitamin C in vitreous humour. We conclude, therefore, that vitreous humour contains factors important for the regulation of prostaglandin metabolism in the eye.     

Nanomolar concentrations of Cd2+ inhibit Ca2+ transport systems in plasma membranes and intracellular Ca2+ stores in intestinal epithelium

The interactions of Cd2+ with active Ca2+ transport systems in rat intestinal epithelial cells have been investigated. ATP-driven Ca2+ transport in basolateral plasma membrane vesicles was inhibited by Cd2+ with an I50 value of 1.6 nM free Cd2+ at 1 microM free Ca2+, using EGTA and HEEDTA to buffer Ca2+ and Cd2+ concentrations, respectively. The inhibition was competitive in nature since the Km value of Ca2+ increased with increasing Cd2+ concentrations while the Vmax remained constant. Cd2+ had similar effects on ATP-dependent Ca2+ uptake by permeabilized enterocytes, indicating that non-mitochondrial and mitochondrial Ca2+ stores are also inhibited by nanomolar concentrations of Cd2+. We conclude that ATP-driven Ca2+ transport systems are the most sensitive elements so far reported in Cd2+ intoxication.     

Effects of hypothyroidism on the distribution and fatty acyl composition of phospholipids in sarcoplasmic reticulum of fast skeletal muscle of the rat

The distribution of phospholipids and fatty acyl composition of individual phospholipids in sarcoplasmic reticulum from fast skeletal muscle of hypothyroid and euthyroid (control) rats have been determined. Hypothyroidism resulted in a 24% decrease in the phosphatidylethanolamine (PE) content and a concomitant increase in the phosphatidylcholine (PC) content of the sarcoplasmic reticulum. The amounts of other phospholipids and cholesterol remained unaffected. Fatty acyl compositions of PE and PC were quantitatively different, but hypothyroidism affected these compositions similarly. Changes included an increase in the proportions of docosahexaenoic (22:6(n - 3)), arachidonic (20:4(n - 6)), icosatrienoic (20:3(n - 6)) and stearic (18:0) acids and a decrease in those of linoleic (18:2(n - 6)), palmitic (16:0) and oleic (18:1(n - 9)) acids. The effects of hypothyroidism on the phospholipid distribution could be reversed by treatment of hypothyroid animals with thyroid hormone for a period of 14 days (10 micrograms T3/100 g body weight per 2 days). The fatty acyl composition of the phospholipids was also restored to the euthyroid values by this treatment. Exceptions were 18:2 and 22:6 in PE, in which case reversal was significant but not complete, and 18:2, 20:4 and 22:6 in PC. The levels of these acids in PC were not reversed to the euthyroid values after the 14-day treatment, but rather the opposite occurred.     

Isolation and characterization of a collagen fibril-associated dermatan sulphate proteoglycan from bovine lung

Dermatan sulphate proteoglycans have been extracted from bovine lung with 2.0 M CaCl2 and isolated using CsCl density gradient centrifugation, DEAE ion-exchange chromatography, gel chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Ultrastructurally these proteoglycans are specifically associated with collagen fibrils. Dermatan sulphate (Mr 15.10(3)-35.10(3), with a strong prevalence for the higher Mr) is link via an O-glycosidic bond to a protein core, which is rich in Asx, Glx and Leu. Of the total uronic acid, 91% is iduronic acid. A part of the glucuronic acid residues is located near the protein core and a large cluster of disaccharides is devoid of glucuronic acid residues. An inhibition enzyme immunoassay has been developed to quantitate the proteoglycan. A model for the interaction between dermatan sulphate proteoglycans and collagen fibrils is proposed.     

Effect of pH on the kinetics of Na+-dependent phosphate transport in rat renal brush-border membranes

The kinetics of Na+-dependent phosphate uptake in rat renal brush-border membrane vesicles were studied under zero-trans conditions at 37 degrees C and the effect of pH on the kinetic parameters was determined. When the pH was lowered it turned out to be increasingly difficult to estimate initial rates of phosphate uptake due to an increase in aspecific binding of phosphate to the brush border membrane. When EDTA or beta-glycerophosphate was added to the uptake medium this aspecific binding was markedly reduced. At pH 6.8, initial rates of phosphate uptake were measured between 0.01 and 3.0 mM phosphate in the presence of 100 mM Na+. Kinetic analysis resulted in a non-linear Eadie-Hofstee plot, compatible with two modes of transport: one major low-affinity system (Km approximately equal to 1.3 mM), high-capacity system (Vmax approximately equal to 1.1 nmol/s per mg protein) and one minor high-affinity (Km approximately equal to 0.03 mM), low-capacity system (Vmax approximately equal to 0.04 nmol/s per mg protein). Na+-dependent phosphate uptake studied far from initial rate conditions i.e. at 15 s, frequently observed in the literature, led to a dramatic decrease in the Vmax of the low-affinity system. When both the extra- and intravesicular pH were increased from 6.2 to 8.5, the Km value of the low-affinity system increased, but when divalent phosphate is considered to be the sole substrate for the low-affinity system then the Km value is no longer pH dependent. In contrast, the Km value of the high-affinity system was not influenced by pH but the Vmax decreased dramatically when the pH is lowered from 8.5 to 6.2. These results suggest that the low-affinity, high-capacity system transports divalent divalent phosphate only while the high-affinity, low-capacity system may transport univalent as well as divalent phosphate. Raising medium sodium concentration from 100 to 250 mM increased Na+-dependent phosphate uptake significantly but the pH dependence of the phosphate transport was not influenced. This observation makes it rather unlikely that pH changes only affect the Na+ site of the Na+-dependent phosphate transport system.     

Three-dimensional reconstruction of a human metaphase chromosome from electron micrographs

A complete human metaphase chromosome has been reconstructed from a series of electron microscopical projections obtained by tilting the specimen stage at 3 degree intervals from –60 to +60 degrees. The reconstructed structure is about 3.0 m long, 1.6 m wide, and 0.8 m thick. The mass distribution was fairly homogeneous within the chromatids and neither a hollow nor a dense core was observed. The distribution and course of fibers observed are most consistent with a looping model of chromosome structure.