Immunocytochemical identification of the human α2-macrogiobulin receptor in monocytes and fibroblasts: Monoclonal antibodies define the receptor as a monocyte differentiation antigen

The α₂-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-α₂-macroglobulin receptor monoclonal antibodies bind to 90–100% of the blood monocyte population and not to other blood cells. This defines the α₂-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18–33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.     

Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine,carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT₂ by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of -butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to -butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.Abbreviations TLC thin-layer chromatography     

Diagnosis of Hunter's syndrome carriers; Radioactive sulphate incorporation into fibroblasts in the presence of fructose 1-phosphate

Summary Mutual correction of co-cultivated fibroblasts from patients with Hunter's and Hurler's syndrome could be inhibited by either fructose 1-phosphate or mannose 6-phosphate. In the presence of fructose 1-phosphate a 50% mixture of fibroblasts from a patient with Hunter's syndrome and a normal homozygous individual showed an increased³⁵S-sulphate incorporation into acid mucopolysaccharides. When fibroblast cultures from one obligate and two possible carriers of Hunter's syndrome were tested for³⁵S-sulphate incorporation, the cultures showed either twice the normal³⁵S-sulphate incorporation into acid mucopolysaccharides in the presence of fructose 1-phosphate or an abnormally high incorporation in the presence as well as in the absence of the sugar phosphate.     

Inhibition of cyclic amp phosphodiesterase by 5'-deoxy-5'-S-isobutylthioadenosine at biologically active concentrations of drug

5'-Deoxy-5'-S-isobutylthioadenosine (SIBA), a synthetic analogue of S-adenosylhomocysteine, has been reported by others to inhibit a number of biological processes and these effects of SIBA have been attributed generally to inhibition of methyltransferases. However, the present studies with mouse lymphocytes show that SIBA also acts as a competitive inhibitor (K_i = 130 μM) of the high-affinity cyclic AMP phosphodiesterase and potentiates the cyclic AMP response of intact cells to several activators of adenylate cyclase. Moreover, SIBA has been found to inhibit lymphocyte-mediated cytolysis, a cellular function known to be sensitive to elevated lymphocyte levels of cyclic AMP, at concentrations (IC₅₀ = 250 μM) similar to those which inhibit cyclic AMP phosphodiesterase. These results indicate the need for caution in attributing biological effects of SIBA singularly to inhibition of methyltransferases and suggest the possible agency of cyclic AMP in the mechanism of SIBA action.     

H-Y antigen in transsexuality,and how to explain testis differentiation in H-Y antigen-negative males and ovary differentiation in H-Y antigen-positive females

Summary H-Y antigen was determined in eight transsexual patients. Two of the four male-to-female transsexual patients typed as H-Y antigen-negative, while the other two typed as expected from their phenotypic and gonadal sex, namely H-Y antigen-positive. Of the four female-to-male transsexual patients, three typed as H-Y antigen-positive and one was H-Y antigen-negative, as expected. The presence of normal testes in H-Y antigen-negative males is assumed to result from a mutation of nucleotide sequences of the H-Y structural gene for antigenic determinants. Thus, an H-Y is produced with normal receptor-binding activity which can sustain the testis determination of the bipotent gonadal anlage. In the case of H-Y antigen-positive females with normal ovaries a deletion of the autosomally located H-Y structural gene is assumed. This deletion should affect sequences for repressor-binding (as was suggested for H-Y antigen-positive XX-males) and for receptor-binding activity of the H-Y antigen molecule. The resulting H-Y antigen is unable to bind to the gonadal receptor of the bipotent gonadal anlage. Thus an ovary is determined. The relevance of H-Y antigen for the aetiology of transsexualism is discussed.     

Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Rat liver fructose-1,6-bisphosphatase, which was assayed by measuring the release of 32P from fructose 1,6-[1-32P]bisphosphate at pH 7.5, exhibited hyperbolic kinetics with regard to its substrate. beta-D-Fructose 2,6-bisphosphate, an activator of hepatic phosphofructokinase, was found to be a potent inhibitor of the enzyme. The inhibition was competitive in nature and the Ki was estimated to be 0.5 microM. The Hill coefficient for the reaction was 1.0 in the presence and absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate also enhanced inhibition of the enzyme by the allosteric inhibitor AMP. The possible role of fructose 2,6-bisphosphate in the regulation of substrate cycling at the fructose-1,6-bisphosphatase step is discussed.     

Fructose 2,6-bisphosphate. A new activator of phosphofructokinase

A new activator of rat liver phosphofructokinase was partially purified from rat hepatocyte extracts by DEAE-Sephadex chromatography. The activator, which eluted in the sugar diphosphate region, was sensitive to acid treatment but resistant to heating in alkali. Mild acid hydrolysis resulted in the appearance of a sugar monophosphate which was identified as fructose 6-phosphate by gas chromatography/mass spectroscopy. These observations suggest that the activator is fructose 2,6-bisphosphate. This compound was synthesized by first reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide and then treating the cyclic intermediate with alkali. The structure of the synthetic compound was definitively identified as fructose 2,6-bisphosphate by 13C NMR spectroscopy. Fructose 2,6-bisphosphate had properties identical with those of the activator purified from hepatocyte extracts. It activated both the rat liver and rabbit skeletal muscle enzyme in the 0.1 microM range and was several orders of magnitude more effective than fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate was not a substrate for aldolase or fructose 1,6-bisphosphatase. It is likely that this new activator is an important physiologic factor of phosphofructokinase in vivo.     

A Simple Device for Staining Many Single-Hole Grids Without Individual Manipulation

In serial sectioning for electron microscopy one of the greatest problems encountered is that the Formvar support film may break when grids are being mounted in or removed from a holder used for staining, or during staining. The latter is particularly troublesome when grids are stained individually. We describe here a device that conveniently eliminates this problem.     

Intracytoplasmic membrane formation and increased oxidation of glycerol growth of Gluconobacter oxydans.

Gluconobacter oxydans is well known for the limited oxidation of compounds and rapid excretion of industrially important oxidation products. The dehydrogenases responsible for these oxidations are reportedly bound to the cell's plasma membrane. This report demonstrates that fully viable G. oxydans differentiates at the end of exponential growth by forming dense regions at the end of each cell observed with the light microscope. When these cells were thin sectioned, their polar regions contained accumulations of intracytoplasmic membranes and ribosomes not found in undifferentiated exponentially growing cells. Both freeze-fracture-etched whole cells and thin sections through broken-cell envelopes of differentiated cells demonstrate that intracytoplasmic membranes occur as a polar accumulation of vesicles that are attached to the plasma membrane. When cells were tested for the activity of the plasma membrane-associated glycerol dehydrogenase, those containing intracytoplasmic membranes were 100% more active than cells lacking these membranes. These results suggest that intracytoplasmic membranes are formed by continued plasma membrane synthesis at the end of active cell division.