Search

Prostaglandin E1 (PGE1) may relieve rest pain and heal ulcers in critical limb ischemia, but its mechanism of action is still incompletely understood. To investigate the effects of PGE1 treatment on endothelial function evaluated as brachial artery flow-mediated vasodilation (FMV) and on soluble adhesion molecule plasma levels (vascular adhesion molecule-1 [sVCAM-1] and intercellular adhesion molecule-1 [sICAM-1]), 12 patients with critical limb ischemia were treated with daily PGE IV infusion (alprostadil 60 microg) for 2 weeks. FMV and plasma sICAM-1 and sVCAM-1 concentrations were determined at baseline, after the first infusion, and after 1 and 2 weeks. Compared with 30 healthy control subjects, patients had higher baseline sVCAM-1 (2.402 +/- 296 ng/ml vs 972 +/- 117 ng/ml) and sICAM-1 levels (464 +/- 51 ng/ml vs 206 +/- 37 ng/ml, both p < 0.05) and lower FMV (1.0 +/- 1.1% vs 5.6 +/- 1.6%, p < 0.05). sICAM-1 concentration progressively decreased with treatment (from 464 +/- 51 ng/ml to 326 +/- 56 ng/ml, 288 +/- 42 ng/ml, and 279 +/- 44 ng/ml after the first dose and, respectively, after 1 and 2 weeks; all p < 0.05). sVCAM-1 showed a reduction after 2 weeks (from 2.402 +/- 296 ng/ml to 1.916 +/- 176 ng/ml; p < 0.05). FMV improved after 1 and 2 weeks (from 1.0 +/- 1.1% to 3.1 +/- 0.6% and 5.2 +/- 2.1%, both p < 0.05). In conclusion, treatment with PGE1 determines a significant improvement in endothelial function in patients with critical limb ischemia.

A case of sudden death in an old female carrier of Duchenne muscular dystrophy is reported. Typical electrocardiographic features were registered without other signs or symptoms of heart involvement. In particular, cardiomyopathy was excluded by echocardiographic evaluation. We believe that in case of a typical electrocardiogram of Duchenne muscular dystrophy, a careful family medical history should be undertaken to exclude a carrier condition.

Objectives: We sought to assess the prognostic value of alterations in left ventricular (LV) diastolic function in patients with essential hypertension., Background: Alterations in LV diastolic function are frequent in patients with hypertension, even in the absence of LV hypertrophy, but their prognostic significance has never been investigated., Methods: In the setting of the Progetto Ipertensione Umbria Monitoraggio Ambulatoriale (PIUMA) study, we followed, for up to 11 years (mean: 4.4 years), 1,839 Caucasian hypertensive patients (50 +/- 12 years, 53% men, blood pressure (BP) 156/98 mm Hg) without previous cardiovascular events, who underwent Doppler echocardiography and 24-h BP monitoring before therapy. The early/atrial (E/A) mitral flow velocity ratio was calculated and corrected for age and heart rate (HR)., Results: During follow-up, there were 164 major cardiovascular events (2.04 per 100 patient-years). The incidence of cardiovascular events was 2.47 and 1.65 per 100 patient-years in patients with an age- and HR-adjusted E/A ratio below (n = 919) and above (n = 920) the median value, respectively (p < 0.005 by the log-rank test). In Cox analysis, controlling for age, gender, diabetes, cholesterol, smoking, LV mass and 24-h systolic BP (all p < 0.05), a low age- and HR-adjusted E/A ratio conferred an increased risk of cardiovascular events (odds ratio 1.57, 95% confidence interval [CI] 1.11 to 2.18, p < 0.01). A 21% excess risk was found for each 0.3 decrease of the adjusted E/A ratio (95% CI from +2% to +43%; p = 0.03)., Conclusions: Impaired LV early diastolic relaxation, detected by pulsed Doppler echocardiography, identifies hypertensive patients at increased cardiovascular risk. Such association is independent of LV mass and ambulatory BP.

Methods: We evaluated the postprandial lipid metabolism in patients with normolipemic peripheral arterial disease (PAD) after the administration of an oral fat load. Total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), HDL2 and HDL3 subfractions, triglycerides (TGs), lipoprotein(a), and LDL size were determined at baseline and for 8 hours after the meal., Results: In patients with PAD, TGs increased significantly at the 4th, 6th, and 8th hours postprandially; in control subjects, TGs increased at the 4th and 6th hours. HDL decreased significantly at the 4th, 6th, and 8th hours in patients with PAD and at the 6th hour in control subjects. The magnitude of postprandial lipemia, expressed as "the area under the incremental curve for TGs," was higher in patients with PAD than in control subjects (770 +/- 476 vs 391 +/- 195 mg/dL at 8 hours, P <.05). Multiple-regression analysis showed that baseline TGs were positively related to the magnitude of postprandial lipemia (P =.01) and that LDL size was negatively related (P =.05)., Conclusions: This is the first documentation of postprandial behavior in patients with normolipemic PAD, suggesting the relevance of postprandial lipoprotein metabolism in the pathogenesis of peripheral atherosclerosis.

Background/aim: Postmenopausal age is characterized by a higher risk for coronary heart disease (CHD) and postprandial lipemia is strictly related with the evidence of CHD. The aim of the study was to clarify the vascular effects of postprandial state in postmenopausal women., Methods: Ten postmenopausal women (mean age 57 +/- 8 years) without vascular risk factors and history of cardiovascular disease underwent an oral fat load test. Endothelial function, expressed as brachial flow-mediated vasodilation (FMV), lipid parameters and reduced glutathione (GSH) were evaluated at baseline and 2, 4 and 6 h after the load., Results: FMV showed a significant decrease at the 2nd hour (2.3 +/- 2.6%, vs. baseline 7.7 +/- 2.8%, p < 0.05) and overlapping to the basal value after 4 h. Triglycerides increased postprandially at the 2nd and 4th hour (1.6 +/- 0.6 micromol/l, 1.8 +/- 0.5 micromol/l vs. baseline 0.9 +/- 0.4 micromol/l, p < 0.05), decreasing thereafter. GSH decreased at the 2nd hour of the postprandial phase (5.1 +/- 1.9 micromol/l vs. baseline 8.4 +/- 1.9 micromol/l, p < 0.05), normalizing successively. At the univariate analysis a negative correlation was found between FMV and triglyceride changes (r = -0.37, p < 0.05) and a positive one between FMV and GSH modifications (r = 0.40, p < 0.05)., Conclusion: These data demonstrated that postprandial lipemia transiently impairs endothelial reactivity by an oxidative burden, partly dependent to triglyceride increase., (Copyright 2002 S. Karger AG, Basel)

Background: Endothelial dysfunction is considered the earliest stage of atherosclerosis. Postprandial phase is associated with a transient impairment of endothelial function concomitantly with the triglyceride-rich lipoprotein increase. This phenomenon may be explained by the oxidative burden induced by triglyceride-rich lipoproteins, reducing nitric oxide bioavailability., Objective: To investigate the effect of a diet enriched with L-arginine, the substrate for nitric oxide synthesis on endothelial function in healthy volunteers., Methods: Endothelial function (expressed as flow-mediated vasodilation (FMV) of the brachial artery), total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, LDL-size, Lp (a) and reduced glutathione (GSH) were evaluated in seven healthy males (mean age 23 +/- 3 years) without cardiovascular risk factors. Measurements were made at baseline and 2, 4 and 6 h after a standardized oral fat load. L-arginine (6 g daily) was administered for 10 days. On the 11th day the oral fat load and the parameters examined previously at entry were repeated., Results: After the first oral fat load, FMV significantly decreased at 2 and 4 h, and overlapped with the basal levels at 6 h. After L-arginine treatment, FMV significantly decreased at 2 h and normalized after 4 and 6 h. Triglycerides increased at 2 and 4 h and decreased after 6 h in both sets of observations relating to before and after L-arginine administration. GSH dropped 2 h after the fat load, both before and after L-arginine. Before L-arginine, FMV exhibited a significant correlation with triglycerides (r= -0.426, P= 0.024) and GSH (r=0.48; P=0.009). After L-arginine, FMV was related to GSH (r=0.39; P=0.03) but not to triglycerides (r= -0.12; P=0.52)., Conclusion: Postprandial endothelial impairment is partly abolished by L-arginine administration. These data, which require confirmation, suggest the importance of dietary choice for atherosclerosis prevention even in young healthy subjects.

Large artery intima-media thickness (IMT) is considered an integrated marker for the total individual burden of arteriosclerosis, and a graded index for cardiovascular risk. However, several different aggregate indexes of IMT on B-mode ultrasound have been used by various investigators, and the optimal number of IMT readings is currently unsettled. In 128 newly diagnosed, never treated, uncomplicated hypertensive subjects aged <55 years (43 +/- 9 years, blood pressure [BP] 152/99 mm Hg), we measured left ventricular mass (M-mode echocardiography, average of five or more measurements) and IMT of common carotid and common femoral arteries. For each segment, 12 IMT measurements were performed, and the average of 1 and 3 readings (right far wall), 6 readings (right side), and 12 readings (right and left side, far and near wall, 3 sampling points) was analyzed. The relation of IMT with left ventricular mass increased progressively with increasing number of readings, from 0.35 (1 reading) to 0.51 (12 readings) for common carotid artery, and from 0.31 to 0.56 for common femoral artery (both P <.001). For each 0.2-mm increase in common femoral IMT, the age-adjusted relative risk of having left ventricular hypertrophy was 1.31 for 1 reading, and increased up to 3.59 for the average of 12 readings. In summary, the association of IMT with left ventricular mass depends strongly on the number of IMT readings. The average of several readings in each segment, including right and left side and far and near wall, carries the closest association to left ventricular mass, and should be preferred for clinical purposes in hypertensive subjects.

The increased risk for coronary artery disease observed in postmenopausal women is partly explained by a more atherogenic fasting lipoprotein profile. Moreover, natural menopause has been associated with an altered postprandial lipid profile. To better characterize the interaction between fasting and postprandial lipid profile after menopause, we examined postprandial changes in several lipid parameters in three age-matched groups of postmenopausal women (16 affected by mixed hyperlipemia, 17 by common hypercholesterolemia, and 17 normolipemic), who underwent a standardized oral fat-loading test. The magnitude of postprandial lipemia, expressed as 8-hour triglyceride incremental area under the curve, was greater in women with mixed hyperlipemia (1,326 +/- 372 mg x dL(-1) x h(-1)) than in normal (484 +/- 384 mg x dL(-1) x h(-1)) and hypercholesterolemic (473 +/- 223 mg x dL(-1) x h(-1); both P <.0001) women, and the differences held after adjustment for body mass index and fasting insulin. Women with mixed hyperlipemia showed a significant postprandial decrease in high-density lipoprotein 2 (HDL(2)) cholesterol, lipoprotein (a), and low-density lipoprotein (LDL) particle size. Both hypercholesterolemic and normolipemic women showed a significant postprandial decrease in HDL cholesterol and lipoprotein (a) levels but not in LDL size. In a multiple linear regression analysis, fasting triglyceride levels, insulin level, and waist-hip ratio were all independent predictors of the magnitude of postprandial lipemia. In conclusion, postmenopausal women with mixed hyperlipemia show a greater postprandial triglyceride increase and a more pronounced reduction in HDL cholesterol level and LDL size than hypercholesterolemic and normolipemic subjects. The presence of the features of insulin resistance syndrome could contribute to the deterioration of postprandial lipemic response in these subjects., (Copyright 2001 by W.B. Saunders Company)

Endothelial dysfunction represents the earliest stage of atherosclerosis and is usually present in hypercholesterolemia. Treatment with statins has been shown to normalize endothelial function in middle-aged men with hypercholesterolemia. We evaluated the effect over time of atorvastatin on the endothelial reactivity in postmenopausal hypercholesterolemic women (mean age, 58 +/- 6 years), receiving atorvastatin, 10 mg daily (n = 20) or American Heart Association step 1 diet (n = 10) for 8 weeks. Lipid profile and brachial artery flow-mediated vasodilation (FMV) were determined at baseline and after 1, 2, 4, and 8 weeks. FMV increased progressively in subjects treated with atorvastatin, and the difference was significant (p < 0.05 vs. baseline) after the second week (baseline 3.8 +/- 3%; first week, 4.8 +/- 3%; second week, 9.2 +/- 3%; fourth week, 11.0 +/- 3%; eighth week, 11.7 +/- 3%). No significant changes were observed in subjects receiving diet (baseline, 3.1 +/- 4%; first week, 2.4 +/- 2%; second week, 2.9 +/- 2%; fourth week, 3.1 +/- 2%; eighth week, 3.3 +/- 2%; p = NS). In the atorvastatin group, low-density lipoprotein (LDL) cholesterol showed a significant decrease since the first week (baseline, 228 +/- 37 mg/dl; first week, 171 +/- 32; second week, 147 +/- 27; fourth week, 139 +/- 29; eighth week, 135 +/- 27; all p < 0.05). In the control group, LDL cholesterol showed a smaller but significant (p < 0.05) reduction after the second week (baseline, 226 +/- 17 mg/dl; first week, 225 +/- 16; second week, 220 +/- 17; fourth week, 203 +/- 27; eighth week, 198 +/- 27). In conclusion, hypercholesterolemic women treated with atorvastatin show a significant improvement in endothelial reactivity after as early as 2 weeks of therapy. The extent to which these beneficial effects are attributable to cholesterol reduction or to a direct effect of the drug remains to be established.

Background: Endothelial dysfunction is considered one of the earliest events in the process of atherosclerosis, and an impaired vasodilatory response has been reported in patients with dyslipidemias. However, the independent association between hypertriglyceridemia and endothelial dysfunction is controversial, and the relation between endothelium-dependent vasodilation and circulating cell adhesion molecules as markers of endothelial dysfunction has not been fully determined., Methods: Brachial artery flow mediated vasodilation (FMV) and the soluble forms of vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) were determined after overnight fasting in 16 men with hypertriglyceridemia (age 33 +/- 6 years) and in 16 age-matched healthy men with normal triglycerides and cholesterol. Subjects who smoked and those with known cardiovascular disease, diabetes, hypertension, recent or active infections, or any other disease that could affect leukocyte activation were excluded from the study., Results: Compared with normal subjects, subjects with hypertriglyceridemia showed a higher level of sVCAM-1 and sICAM-1 (both P <.001), a reduced FMV (P <.01), and a smaller LDL particle size (P <.05). FMV had a significant inverse correlation with sVCAM-1 (r = -0.61, P <.001) and sICAM-1 (r = -0.38, P <.03). LDL particle size had a strong, direct association with FMV (r = 0.75, P <.001) and an inverse association with adhesion molecules. By multiple regression analysis, triglycerides (P <.001) and small LDL particle size (P <.002) predicted a reduced FMV., Conclusions: Serum level of cell adhesion molecules is increased and FMV is impaired in young healthy men with hypertriglyceridemia compared with age-matched men with normal lipid levels. Small, dense LDL particles may play a role in determining endothelial dysfunction in these subjects.

Nondominant hereditary spherocytosis (ndHS) is a disorder characterized in some patients by severe hemolytic anemia and marked deficiency of erythrocyte spectrin. This report describes the identification of a variant spectrin chain, alpha-spectrin Bughill or alpha(BH), that is associated with this disorder in a number of patients. Tryptic maps of spectrin from affected individuals revealed an acidic shift in isoelectric point of the alphaII domain peptides at 46 kD and 35 kD. A point mutation at codon 970 of the alpha-spectrin gene (GCT-->GAT), that changes the encoded amino acid from an alanine to an aspartic acid, was identified in genomic DNA of affected patients. The alpha(BH) variant was present in 8 patients with ndHS from five different kindreds but was absent in 4 patients from two other kindreds. The 8 ndHS patients with the alpha(BH) variant appeared to be homozygous for the alpha(BH) variant by analysis of peptide maps of limited tryptic digests of erythrocyte spectrin. However, following genomic DNA analysis, only 2 of these patients were true homozygotes, whereas 6 were found to be doubly heterozygous for the alpha(BH) allele and a second, presumably abnormal, alpha-spectrin gene. These results suggest that, in these 6 patients, the second alpha-spectrin allele is in fact associated with one or more genetic defect(s), causing decreased accumulation of alpha-spectrin. The pattern of transmission of the alpha(BH) allele in certain families suggests that the alpha(BH) amino-acid substitution is not itself responsible for ndHS but is more likely a polymorphic variant that, in some but not all cases, is in linkage disequilibrium with another uncharacterized alpha-spectrin gene defect that itself is a cause of ndHS.

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are inherited disorders of erythrocyte shape that are frequently associated with abnormalities in alpha-spectrin, one of the principal structural proteins of the erythrocyte membrane skeleton. Five polymorphisms of the alpha-spectrin gene, located in a 6-kb interval of genomic DNA, were identified and analyzed in normal and mutant alpha-spectrin alleles. Three of these polymorphisms are due to single nucleotide substitutions in the alpha-spectrin gene coding region that lead to changes in the amino acid sequence. In combination, these three polymorphisms are responsible for the different peptide phenotypes of the alphaII domain previously observed following limited tryptic digestion of spectrin protein. The most common haplotype, type 1, was found predominantly in Caucasians and was the only haplotype identified in Asians. Haplotypes 2, 3, and 4 were identified predominantly in individuals of African ancestry and were commonly found in patients with HE or HPP. Analysis of coinheritance of alphaII domain polymorphisms with alpha-spectrin gene mutations causing HE or HPP in African-American patients with HE and HPP suggests that, with one exception, a given HE/HPP mutation is present in an alpha-spectrin gene of only one haplotype, indicating a founder effect. The other two polymorphisms located in this region of the alpha-spectrin gene do not change the amino acid sequence of the encoded alpha-spectrin chain and are not in linkage disequilibrium with three of the four alphaII domain haplotypes. A model is proposed for the evolutionary origin of the different haplotypes.

Hereditary elliptocytosis (HE) is a heterogeneous disorder of red blood cells frequently associated with abnormal limited tryptic digestion of the alpha I domain of spectrin and impaired spectrin dimer self-association. We studied two related individuals with poikilocytic hereditary elliptocytosis (HE) of different severity. Limited tryptic digestion of spectrin from these individuals showed the presence of a variant alpha I/50b Kd peptide at the expense of the normal alpha I/80 Kd peptide. Amino acid sequence analysis of the abnormal peptide showed that the proteolytic cleavage occurred after the arginine at position 470 of the alpha spectrin chain. Spectrin from these patients had an impaired ability to undergo self-association, as evidenced by increased amounts of spectrin dimers in 4 degrees C extracts of erythrocyte membrane from affected individuals. The polymerase chain reaction was used to study the DNA sequence of the alpha spectrin gene encoding the region of the alpha spectrin chain surrounding the abnormal proteolytic cleavage site. We detected the in-frame deletion of the trinucleotide CAT, encoding histidine 469, two amino acid residues to the N-terminal side of the abnormal proteolytic cleavage site between residues 470 and 471. Similar to many other defects of spectrin associated with HE, this deletion occurs in helix three of repeat 5 of the proposed triple helical model of spectrin repeats.

Hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis are closely related, congenital disorders of the red blood cell usually associated with defective spectrin self-association and abnormal limited tryptic digestion of the N-terminal of domain of spectrin. Enhanced cleavage by trypsin of spectrin from affected individuals at arginyl residue 45* and lysyl residue 48* frequently yields increased amounts of an alpha 1/74-Kd fragment at the expense of the normal alpha 1/80-Kd parent fragment. Limited tryptic digestion of three unrelated individuals with HPP showed the alpha 1/74 defect. To ascertain the molecular defect responsible for the abnormality, the structure of exon 2 of the alpha-spectrin gene was examined. Genomic DNA from the subjects was amplified by the polymerase chain reaction using primers flanking exon 2. Restriction endonuclease digestion of amplified products showed the loss of the HindIII site at codons 47 and 48 in one allele of subject 1 and abolished the AhaII site at codons 27 and 28 in one allele of subjects 2 and 3. Nucleotide sequence analysis of subcloned amplified DNA from the HPP subjects showed three novel amino acid substitutions. In subject 1 (a black individual), a single base substitution (AAG----AGG) at codon position 48 changes amino acid residue lysine to arginine. In subject 2 (a white individual), a single base substitution (CGT----AGT) at codon 28 changes arginine to serine. In subject 3 (a black individual), a different base substitution at position 28 (CGT----CTT) changes arginine to leucine. These mutations occur at positions of the alpha l domain where other mutations have also been described, indicating that the normal residues at these positions play an important role in spectrin dimer self-association and thus, in membrane stability.

Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.

Human platelet glycoproteins were isolated from whole platelets by two methods. The first method, that of affinity chromatography on wheat germ agglutinin, is based on the known affinity of lectins for cell surface glycoproteins. When solubilized whole platelets are used as starting material for this procedure, elution with N-acetylglucosamine yields primarily a glycoprotein of Mr approximately 150 000 as estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The second method is based on the ability of the chaotropic salt lithium diiodosalicylate to extract glycoprotein from particulate cell fractions in water-soluble form. This method yields three major glycopeptides with apparent molecular weights after sulfhydryl reduction of 145 000, 125 000, and 95 000 as estimated on 5.6% sodium dodecyl sulfate-acrylamide gels. Carboxymethylation of these preparations in the presence of sulfhydryl-reducing agent further resolves a glycoprotein of Mr approximately 165 000. Treatment of whole platelets by periodate oxidation and sodium[3H]-borohydride reduction labels the three major glycoproteins extracted by lithium diiodosalicylate and the glycoprotein of Mr approximately 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with Mr approximately 165 000 resolved by carboxymethylation of the lithium diiodosalicylate extracted glycoprotein mixture was not labelled by this method, suggesting that it represents the granule protein with similar electrophoretic characteristics described by others. Phosphorylation of intact platelets with 32Pi also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the bilipid layer of the platelet membrane, bearing reactive groups on both outer and cytoplasmic surfaces.

A case of postabortal hemorrhage in a patient with von Willebrand's disease was controlled by arteriographic embolization of the uterine branch of the internal iliac artery. Selective internal iliac artery embolization has been effectively used to control postpartum hemorrhage and hemorrhage resulting from pelvic malignancy. A discussion is presented for expanding the use of selective embolization for controlling uterine hemorrhage to patients suffering from postabortal bleeding as a means of controlling hemorrhage without sacrificing fertility.

An abnormal alpha subunit of erythrocyte spectrin has been described in hereditary pyropoikilocytosis (HPP), a rare hemolytic anemia characterized by erythrocyte budding and fragmentation. In HPP spectrin, the N terminal domain of the alpha subunit (alpha I T80) shows increased susceptibility to tryptic digestion, resulting in cleavage to a 50,000-d peptide, presumably due to a change in primary structure of the alpha I domain which alters conformation and generates the new cleavage site. The functional result of this conformational alteration is marked impairment of spectrin oligomer formation in vitro, consistent with the established role of alpha I T80 in spectrin self-association. In the present study, we demonstrate an abnormal spectrin alpha subunit in two kindreds with hereditary elliptocytosis (HE) that is qualitatively identical to HPP spectrin. Clinical expression of HE in these families ranges from mild elliptocytosis without hemolysis to severe poikilocytic hemolytic anemia clinically resembling HPP. In all affected individuals, a fraction of alpha I T80 is abnormal, as shown by its cleavage during mild tryptic digestion to the 50 kd peptide described in HPP; the fraction of alpha I T80 affected is directly proportional to the severity of clinical expression of HE. Spectrin oligomer formation is likewise impaired to a degree which correlates with hematologic disease. One of the HE kindreds studied demonstrated polymorphism in the spectrin alpha II domain, previously described as a frequent occurrence in blacks. This family also demonstrates a variant alpha III domain in spectrin that has not previously been described. We conclude that the abnormality in the alpha I domain originally described in HPP spectrin is shared by a subset of patients with HE; the severity of clinical expression, ranging from mild nonhemolytic HE to poikilocytic hemolytic anemia, is related to the fractional quantity of the alpha subunit that is affected.

A number of proteins that make up the membrane skeleton have been identified, and we have a rough but tantalizing picture of the ways in which they interact to maintain its integrity. An approximate molecular model of spectrin has been proposed, and many new analytic procedures have been developed to search for biochemical variants that may be related to membrane defects. Even at this rudimentary stage of molecular description we have been able to identify structural changes in spectrin that are correlated with pathophysiologic states. The prospects for some genuine insights into the pathogenesis of some congenital hemolytic anemias seem good.

Factor VIII (antihemophilic globulin) has been prepared from Hyland method IV AHG and cryoprecipitate using limited chymotryptic digestion followed by Sepharose gel filtration. The activity of factor VIII is unaffected by the digestion procedure, while fibrinogen in converted to large noncoagulable fragments. The purified factor VIII has been found to be a macromolecular glycoprotein with a major subunit of 240,000, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Carbohydrate analysis of factor VIII gave values of 1% sialic acid, 2.8% hexosamine, and 1-2% hexose (mannose, galactose, and fucose). The lipid content was found to be less than 5% of the protein content, and included no detectable phospholipid. The amino acid content is also reported. Immunoelectrophoretic analysis using rabbit antibody to purified factor VIII produced a single precipitin line. The chymotrypsin digestion step facilitates the preparation of factor VIII by reducing the viscosity of fibrinogen in the crude starting material, thereby increasing fivefold the quantity of material which can be processed at one time. It also improves markedly the resolution between factor VIII and fibrinogen on gel filtration.

The metabolism of the ribosomal and soluble protein components of Aerobacter aerogenes was examined during its incubation in a Mg(++)-deficient medium. Bacteria were exposed to leucine-H(3) during the exponential growth period preceding Mg(++) starvation, and extracts were prepared after intervals of starvation and were centrifuged through gradients of sucrose to separate ribosomal from soluble proteins. Ribosomal proteins synthesized during the preceding exponential growth were slowly lost from the ribosomes; after 8 hr of starvation, few, if any, sedimented with ribosomes. Losses of total protein, together with the known rate of ribosome decay during Mg(++) starvation, suggested that these ribosomal proteins are ultimately degraded to acid-soluble products and account for all protein lost by the starving cells. These conclusions were supported by studies of Mg(++) starvation in a uracil-requiring strain of A. aerogenes: during uracil starvation a smaller fraction of the proteins synthesized were ribosomal, and the fraction of protein which subsequently decayed during Mg(++) starvation was correspondingly less. During recovery from Mg(++) starvation, proteins, lost from disintegrated ribosomes, were not detectably reutilized into new particles even before their degradation to acid-soluble products was complete. Synthesis of soluble proteins continued for more than 24 hr of starvation at a rate per milliliter close to 45% of the instantaneous rate per milliliter of the exponentially growing bacteria at the time Mg(++) was removed. This value agreed with that found previously for synthetic rates of deoxyribonucleic acid, transfer ribonucleic acid, and ribosomal ribonucleic acid during starvation relative to rates during exponential growth.