Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain.

Denaturation by guanidine-HCl, urea, or heating was performed on the common isoforms of human apolipoprotein (apo) E (apoE2, apoE3, and apoE4) and their 22-kDa and 10-kDa fragments in order to investigate the effects of the cysteine/arginine interchanges at residues 112 and 158. Previous physical characterization of apoE3 established that apoE contains two domains, the 10-kDa carboxyl-terminal and 22-kDa amino-terminal domains, which unfold independently and exhibit large differences in stability. However, the physical properties of apoE2, apoE3, and apoE4 have not been compared before. Analysis by circular dichroism showed that the different isoforms have identical alpha-helical contents and guanidine-HCl denaturation confirmed that the two domains unfold independently in all three isoforms. However, guanidine-HCl, urea, and thermal denaturation showed differences in stability among the 22-kDa amino-terminal fragments of the apoE isoforms (apoE4 < apoE3 < apoE2). Furthermore, guanidine-HCl denaturation monitored by circular dichroism and fluorescence suggested the presence of a folding intermediate in apoE, most prominently in apoE4. Thus, these studies reveal that the major isoforms of apoE, which are associated with different pathological consequences, exhibit significant differences in stability.

Apolipoprotein E;-low density lipoprotein receptor interaction. Influences of basic residue and amphipathic alpha-helix organization in the ligand.

Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.

Structure and function of procollagen C-proteinase (mTolloid) domains determined by protease digestion, circular dichroism, binding to procollagen type I, and computer modeling.

Procollagen C-proteinase-2 (pCP-2, mTld) is derived from the longest splicing variant of the gene encoding bone morphogenetic protein 1 (BMP-1). The variants have identical amino terminal signal peptides, prodomains and astacin-like protease domains. However, they differ in the length of their carboxy terminal part, which in pCP-2 has the composition CUB1, CUB2, EGF-like1, CUB3, EGF-like2, CUB4, CUB5, and C-tail. In the shorter form, pCP-1 (i.e., BMP-1), the sequence ends after the CUB3-domain. Using a combination of mutagenesis and structural approaches, we have investigated the structure and function of subfragments of pCP-2. The full-length latent recombinant enzyme and its N-terminally truncated form lacking the prodomain were tested for their enzymic activity. The intact protein showed only partial processing of procollagen type I, whereas the truncated form expressed enzymic activity indistinguishable from its native counterpart purified from chick embryo tendons. These results clearly demonstrated that the prodomain is required for the latency of the enzyme but not for its correct folding. Limited proteolysis of the recombinant protein with alpha-chymotrypsin produced four discrete fragments revealing the location of cleavage sites between the repetitive CUB/EGF domains. The results provide evidence that the CUB sequences form independently folded modules that are stabilized by two pairs of internal disulfide bridges. The modules are linked to each other by more flexible, hinge-like peptides. Solid-phase binding assays with isolated CUB domains and immobilized procollagen type I demonstrated that the first three but not the last two CUB domains specifically bound to the substrate. To define putative sites for CUB-CUB or CUB-substrate interactions, we generated molecular models for pCP-2 CUB domains. The models were obtained using as a template the structure of CUB domain in zona pellucida adhesion protein PSP-I/PSP-II from porcine sperm. The predicted conformations for homology models were, subsequently, confirmed by circular dichroism spectroscopy of polypeptide domains isolated following limited proteolysis with alpha-chymotrypsin.

Umbilical vascular catheters: localization by two-dimensional echocardio/aortography.

Umbilical vascular catheters are often necessary in the care of critically ill neonates. Position of the catheter tip is usually determined by roentgenography. Location of the umbilical arterial or venous catheter was determined by 2-dimensional echocardio/aortography in 55 consecutive infants and was compared to localization by thoraco-abdominal roentgenography. Most of the infants (76%) had respiratory distress syndrome or congenital heart disease. Echocaortographic localization of the umbilical arterial catheter correlated very closely (N = 50, sr = .90) with roentgenographic determination. For localization of the tip of the umbilical venous catheters, echocardiography was more accurate than roentgenography (employing contrast echocardiography for confirmation of cardiac chamber position). Two-dimensional echocardio/aortographic localization of the tip of indwelling umbilical vascular catheters is as accurate as roentgenography in the arterial system and more accurate than x-ray for umbilical venous catheters. Echocardio/aortography is superior to roentgenography (in localizing the catheter tip) because it 1) avoids ionizing radiation, 2) makes positioning of the patient unnecessary, 3) allows visualization of the catheter in relation to cardiovascular structures, and 4) may allow demonstration of intraarterial thrombo-emboli.

Thrombocytopenia and pulmonary hypertension in the perinatal aspiration syndromes.

Thirty-three of 616 consecutively admitted newborn infants had trombocytopenia (platelet count less than 150,00/mm). Only 16 of these were among the 356 infants with lung disease. However, 12 of the 16 were among the 90 infants with a diagnosis of a perinatal aspiration syndrome. The 12 thrombocytopenic infants were the only infants with PAS considered to have pulmonary hypertension. The duration of significant right-to-left shunting of blood paralleled the duration of thrombocytopenia in these infants; PHN was not associated with thrombocytopenia in other neonatal lung diseases. Thus, platelets appear to be important in the pathogenesis of PHN complicating PAS.

The gastric aspirate foam test in the prediction of hyaline membrane disease.

The gastric aspirate foam test was performed on 99 infants of low birth weight. In 59 of 61 infants with no respiratory distress, the test was positive. All infants who developed hyaline membrane disease (HMD) had negative or intermediate foam test results. There was no consistent relationship between the foam test result and the development of wet lung syndrome. Infants who were small for gestational age had a significantly lower incidence of HMD, as did infants with amniotic fluid infection syndrome. These data indicate that the gastric aspirate foam test is a reliable index of neonatal lung maturity.

Serum ferritin estimation in the assessment of iron stores in severe iron deficiency anaemia in pregnancy and the response to treatment.

Serum ferritin estimation was used to determine the iron stores in 55 patients with severe iron deficiency anaemia in pregnancy. The response to oral or intravenous iron therapy was monitored in 18 of these patients. The results in all cases indicated deficient iron stores. Replenishment of iron stores was significantly greater in those patients treated with parenteral iron. This may be the treatment of choice for severe iron deficiency anaemia during pregnancy.

Incidence of hyaline membrane disease in the Cape Coloured.

Hyaline membrane disease (HMD), which carries a significant mortality and morbidity, is usually a disorder of the preterm infant. Several workers have shown that there is a lower incidence in the Black and Coloured neonate than in the White neonate. These studies have not excluded the growth-retarded baby, in whom the functional maturation of vital organs may be accelerated. This article examines the incidence of HMD among appropriately grown Cape Coloured infants and compares this with the incidence in White infants.