Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor.

Ten peptides, derived from human alpha 2-macroglobulin (alpha 2M) receptor by chemical or proteolytic digestion, were sequenced. Comparative analysis revealed that all of the resulting sequences were present within the cDNA-deduced structure of low density lipoprotein receptor-related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988) EMBO J. 7, 4119-4127). The findings provide evidence that the alpha 2M receptor and LRP are the same molecule. Further evidence comes from immunoprecipitation experiments using a monoclonal antibody specific for the alpha 2M receptor that show this molecule, like LRP, to contain two polypeptides of approximately 420 and 85 kDa that are noncovalently associated. An additional component of this receptor system is a 39-kDa polypeptide that co-purifies with the alpha 2M receptor during affinity chromatography. Solid phase binding studies reveal that the 39-kDa polypeptide binds with high affinity (Kd = 18 nM) to the 420-kDa component of the alpha 2M receptor. The apparent identity of LRP and the alpha 2M receptor suggests that this molecule is a multifunctional receptor with the capacity to bind diverse biological ligands and highlights a possible relationship between two previously unrelated biological processes, lipid metabolism and proteinase regulation.     

Adenylyl cyclase deficient cr-1 (Crisp) mutant of Neurospora crassa: Cyclic AMP-dependent nutritional deficiencies

The inability to synthesize cyclic AMP drastically affects the nutritional metabolism of Neurospora crassa. The adenylyl cyclase-less mutant cr-1 (crisp) did not utilize several carbon sources, including glycerol, mannitol, arabinose, and casaminoacids. However, in glucose or acetate it grew as well as the wild type. The following evidence suggested that these nutritional deficiencies were a direct result of the cr-1 mutation: (i), in crosses to wild type they segregated together with the crisp morphological marker; (ii), cyclic AMP added to the cr-1 mutant growth medium overcame the nutritional deficiencies; (iii), the cyclic AMP effect was specific for the crisp mutant, for it was not observed with the wild type, nor with a spontaneous glycerol-utilizing cr-1 strain.     

Trisomy 6p22→6pter due to familial t(6;13)(p22;q34 or 33) translocation

Summary 235 cases of Down's syndrome were ascertained in a 10-year study of Down's syndrome in Western Australia. Although cytogenetic studies performed on 222 subjects confirmed that 95% of cases were trisomic due to nondisjunction, 4% were trisomic due to translocation, and 1% were mosaic, the ratio of inherited/sporadic translocations differed from that usually reported. Comparison of the results with those of an earlier Australian survey of Down's syndrome demonstrated a real fall in the incidence of Down's syndrome in Australia but no significant change in maternal age-specific incidences.     

A case of true hermaphroditism with 45X/46XY mosaicism

Summary The authors describe a case of true hermaphroditism of mainly female phenotype, ambiguous external genitalia, and ovotestis. The cytogenetic studies revealed 45X/46XY mosaicism and an absence of Barr bodies.     

NMR Structural Studies of Domain 1 of Receptor-associated Protein

The 39 kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds tightly to the low-density lipoprotein receptor-related protein (LRP) as well as to other members of the low-density lipoprotein receptor superfamily. The association of RAP with LRP prevents this receptor from interacting with ligands. RAP is a three-domain protein that contains two independent LRP binding sites; one located within domains 1 and 2, and one located within domain 3. As the first step toward defining the structure of the full-length protein and understanding the interaction between RAP and this family of receptors, we have determined the 3D structure of domain 1 using constraints derived from heteronuclear multi-dimensional NMR spectra, including NOEs, dihedral angles, J-couplings and chemical shifts, as well as two sets of non-correlated residual dipolar couplings measured from the protein solutions in anisotropic media of Pf1 and 6% polyacrylamide gel. The backbone C(alpha) rmsd between the current structure and a homo-nuclear NOE-based structure is about 2 A. The large rmsd mainly reflects the significant differences in helical orientation and in the structural details of the long helix (helix 2) between the two structures.     

Glycerokinase activity in brown adipose tissue: a sympathetic regulation?

The effect of brown adipose tissue (BAT) sympathetic hemidenervation on the activity of glycerokinase (GyK) was investigated in different physiological conditions. In rats fed a balanced diet, the activity of the enzyme was approximately 50% lower in BAT-denervated pads than in intact, innervated pads. In rats adapted to a high-protein, carbohydrate-free diet, norepinephrine turnover rates and BAT GyK activity were already reduced, and BAT denervation resulted in a further decrease in the activity of the enzyme. Cold acclimation of normally fed rats at 4 degrees C for 10 days markedly increased the activity of the enzyme. Cold exposure (4 degrees C) for 6 h was insufficient to stimulate BAT GyK, but the activity of the enzyme was already increased after 12 h of cold exposure. The cold-induced BAT GyK stimulation was completely blocked in BAT-denervated pads. The data indicate that an adequate sympathetic flow to BAT is required for the maintenance of normal levels of GyK activity and for the enzyme response to situations, such as cold exposure, which markedly increase BAT sympathetic flow.     

Glyconeogenic pathway in isolated skeletal muscles of rats

Although the conversion of lactate to glycogen (glyconeogenesis) in muscle was demonstrated a long time ago, the biochemical reactions responsible for this process are still a controversial matter. In the present study, advantage was taken from the specific inhibition induced by phenylalanine on muscle pyruvate kinase (PK) to investigate the role of reverse PK activity in muscle glyconeogenesis. Addition of phenylalanine to the incubation medium of a preparation of isolated, intact skeletal muscles that maintain metabolic activity for several hours reduced by 50% the rate of incorporation of [14C]lactate or [14C]bicarbonate into muscle glycogen. Muscle extracts presented high levels of maximal activity of PK in the reverse direction, which was completely blocked in the presence of phenylalanine. In contrast, mercaptopicolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), did not affect the incorporation of 14C from either lactate or bicarbonate into muscle glycogen. Maximal PEPCK activity was much lower in muscle extracts than in gluconeogenic or glyceroneogenic tissues and was suppressed in the presence of mercaptopicolinic acid. The data suggest that a reversal of the metabolic flux through the reaction catalyzed by PK contributes to the accumulation of lactate-derived glycogen that occurs in skeletal muscle under certain physiological conditions.     

Type I collagen contains at least 14 cryptic fibronectin binding sites of similar affinity

There is uncertainty in the literature regarding the number and location of fibronectin binding sites on denatured collagen. Although most attention has focused on a single site near the collagenase-sensitive region of each alpha chain, there is evidence for additional sites in other regions. We treated bovine type I collagen with cyanogen bromide, labeled the resulting mixture with fluorescein, and separated the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent bands were excised from the gel and dialyzed exhaustively to remove detergent. Titration of eight distinct fluorescent-labeled fragments with the 42-kDa gelatin-binding fragment of fibronectin caused increases in anisotropy that were fully reversible with unlabeled gelatin. By fitting the dose responses it was possible to calculate apparent K(d)'s whose values ranged between 1 and 4 microM. The largest fragment, alpha(2)-CB3,5, composing about 2/3 of the alpha(2) chain, when further digested with endoproteinase Lys-C, yielded at least three additional subfragments that also bound with similar affinities. Thus, there appear to be at least 14 distinct fibronectin binding sites of similar affinity in bovine type I collagen, five on each of the alpha(1) chains and four on the alpha(2) chain. Experiments with several synthetic peptides failed to reveal the exact nature of the binding site.     

Catecholamines inhibit Ca(2+)-dependent proteolysis in rat skeletal muscle through beta(2)-adrenoceptors and cAMP

Overall proteolysis and the activity of skeletal muscle proteolytic systems were investigated in rats 1, 2, or 4 days after adrenodemedullation. Adrenodemedullation reduced plasma epinephrine by 95% and norepinephrine by 35% but did not affect muscle norepinephrine content. In soleus and extensor digitorum longus (EDL) muscles, rates of overall proteolysis increased by 15-20% by 2 days after surgery but returned to normal levels after 4 days. The rise in rates of protein degradation was accompanied by an increased activity of Ca(2+)-dependent proteolysis in both muscles, with no significant change in the activity of lysosomal and ATP-dependent proteolytic systems. In vitro rates of Ca(2+)-dependent proteolysis in soleus and EDL from normal rats decreased by ~35% in the presence of either 10(-5) M clenbuterol, a beta(2)-adrenergic agonist, or epinephrine or norepinephrine. In the presence of dibutyryl cAMP, proteolysis was reduced by 62% in soleus and 34% in EDL. The data suggest that catecholamines secreted by the adrenal medulla exert an inhibitory control of Ca(2+)-dependent proteolysis in rat skeletal muscle, mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.     

Glyceroneogenesis and the supply of glycerol-3-phosphate for glyceride-glycerol synthesis in liver slices of fasted and diabetic rats

The pathways of glycerol-3-phosphate (G3P) generation for glyceride synthesis were examined in precision-cut liver slices of fasted and diabetic rats. The incorporation of 5 mM [U-(14)C]glucose into glyceride-glycerol, used to evaluate G3P generation via glycolysis, was reduced by approximately 26-36% in liver slices of fasted and diabetic rats. The glycolytic flux was reduced by approximately 60% in both groups. The incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol (glyceroneogenesis) increased approximately 50% and approximately 36% in slices of fasted and diabetic rats, respectively, which also showed a two-fold increase in the activity phosphoenolpyruvate carboxykinase. The increased incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol by slices of fasted rats was not affected by the addition of 5 mM glucose to the incubation medium. The activity of glycerokinase and the incorporation of 1 mM [U-(14)C]glycerol into glyceride-glycerol, evaluators of G3P formation by direct glycerol phosphorylation, did not differ significantly from controls in slices of the two experimental groups. Rates of incorporation of 1 mM [2-(14)C]pyruvate and [U-(14)C]glycerol into glucose of incubation medium (gluconeogenesis) were approximately 140 and approximately 20% higher in fasted and diabetic slices than in control slices. It could be estimated that glyceroneogenesis by liver slices of fasted rats contributed with approximately 20% of G3P generated for glyceride-glycerol synthesis, the glycolytic pathway with approximately 5%, and direct phosphorylation of glycerol by glycerokinase with approximately 75%. Pyruvate contributed with 54% and glycerol with 46% of gluconeogenesis. The present data indicate that glyceroneogenesis has a significant participation in the generation of G3P needed for the increased glyceride-glycerol synthesis in liver during fasting and diabetes.