An immune tolerance approach using transient low-dose methotrexate in the ERT-naïve setting of patients treated with a therapeutic protein: experience in infantile-onset Pompe disease.

PURPOSE: To investigate immune tolerance induction with transient low-dose methotrexate (TLD-MTX) initiated with recombinant human acid α-glucosidase (rhGAA), in treatment-naïve cross-reactive immunologic material (CRIM)-positive infantile-onset Pompe disease (IOPD) patients. METHODS: Newly diagnosed IOPD patients received subcutaneous or oral 0.4 mg/kg TLD-MTX for 3 cycles (3 doses/cycle) with the first 3 rhGAA infusions. Anti-rhGAA IgG titers, classified as high-sustained (HSAT; ≥51,200, ≥2 times after 6 months), sustained intermediate (SIT; ≥12,800 and <51,200 within 12 months), or low (LT; ≤6400 within 12 months), were compared with those of 37 CRIM-positive IOPD historic comparators receiving rhGAA alone. RESULTS: Fourteen IOPD TLD-MTX recipients at the median age of 3.8 months (range, 0.7-13.5 months) had a median last titer of 150 (range, 0-51,200) at median rhGAA duration ~83 weeks (range, 36-122 weeks). One IOPD patient (7.1%) developed titers in the SIT range and one patient (7.1%) developed titers in the HSAT range. Twelve of the 14 patients (85.7%) that received TLD-MTX remained LT, versus 5/37 HSAT (peak 51,200-409,600), 7/37 SIT (12,800-51,000), and 23/37 LT (200-12,800) among comparators. CONCLUSION: Results of TLD-MTX coinitiated with rhGAA are encouraging and merit a larger longitudinal study.

Bile pigment formation in plants.

The unicellular alga Cyanidium caldarium evolves carbon monoxide during the syntheis of the bile pigment, phycocyanobilin. Carbon monoxide and phycocyanobilin were produced in stoichiometric amounts at comparable rates. Therefore, the mechanism of bile pigment formation in this plant parallels that in mammals.

Safe Sleep Guideline Adherence in Nationwide Marketing of Infant Cribs and Products.

BACKGROUND: Sudden infant death syndrome and sleep-related sudden unexpected infant death remain leading causes of infant mortality in the United States despite 4 safe sleep guideline restatements over the previous 24 years. Advertising and retail crib displays often promote infant sleep environments that are counter to the most recent American Academy of Pediatrics (AAP) guidelines. METHODS: Magazine advertisements featuring sleep in parenting magazines from 1992, 2010, and 2015 were reviewed for adherence. Crib displays from nationwide retailers were surveyed for adherence to the latest AAP safe sleep guidelines. The primary outcome was adherence to the guidelines. RESULTS: Of 1758 retail crib displays reviewed, only half adhered to the latest AAP guidelines. The most common reasons for nonadherence were the use of bumper pads and loose bedding. The depiction of infant cribs and sleep products in magazine advertising has become significantly more adherent over time; however, 35% of current advertisements depict nonadherent, unsafe sleep environments. Magazine advertising portraying safe sleep environments revealed racial and ethnic disparities. CONCLUSIONS: Although improvements have been made over time with increased adherence to AAP safe sleep guidelines, significant deficiencies remain. Advertising continues to depict unsafe sleep environments. Crib manufacturers and retail establishments continue to market and sell bedding and sleep products considered unsafe by the AAP in approximately half of retail crib displays. Pediatric and public health care providers should continue educational and advocacy efforts aimed at the public, but should also include retailers, manufacturers, and advertising professionals to foster improved sleep environments for all children.

Sustained immune tolerance induction in enzyme replacement therapy-treated CRIM-negative patients with infantile Pompe disease.

BACKGROUND: Cross-reactive immunological material-negative (CRIM-negative) infantile Pompe disease (IPD) patients develop an immune response against enzyme replacement therapy (ERT) with alglucosidase alfa that nullifies ERT efficacy. Prophylactic immune tolerance induction (ITI) with rituximab, methotrexate, and IVIG successfully prevents development of deleterious rhGAA IgG antibodies; however, safety, likelihood of success, and long-term efficacy of ITI in a larger cohort remain unknown. METHODS: Clinical data were analyzed for 19 CRIM-negative IPD patients who received ITI with rituximab, methotrexate, and IVIG in the ERT-naive setting (ERT+ITI) and compared to a historical cohort of 10 CRIM-negative IPD patients on ERT monotherapy. RESULTS: ITI was safely tolerated, although infections were reported in 4 patients. Fourteen (74%) ERT+ITI patients were alive, with a median age of 44.2 months at their final assessment. The eldest survivor was 103.9 months old, with 100.2 months of follow-up after initiation of ERT+ITI. Death (n = 5) occurred at a median age of 29.2 months and was unrelated to the administration of ITI. Fifteen patients either did not seroconvert (n = 8) or maintained low titers (n = 7; defined as titers of ≤6,400 throughout the course of ERT) following ERT+ITI. Only one patient developed high and sustained antibody titers (defined as titers of ≥51,200 at or beyond 6 months on ERT). Left ventricular mass index (LVMI) decreased from a median of 248.5 g/m2 at baseline to 76.8 g/m2 at a median time from ERT+ITI initiation to 59 weeks. ERT+ITI significantly improved overall survival (P = 0.001), eliminated/reduced antibodies at values of ≤6,400 at week 52 on ERT (P = 0.0004), and improved LVMI at week 52 on ERT (P = 0.02) when compared with ERT monotherapy. CONCLUSION: Evidence from this international cohort of CRIM-negative IPD patients further supports the safety, feasibility, and efficacy of ITI in the prevention of immune responses to ERT. TRIAL REGISTRATION: Clinicaltrials.gov NCT01665326. FUNDING: This research was supported in part by the Lysosomal Disease Network, a part of NIH Rare Diseases Clinical Research Network, and by a grant from Genzyme, a Sanofi company.

Phycobiliprotein synthesis in protoplasts of the unicellular cyanophyte, Anacystis nidulans.

Stable and metabolically active protoplasts were prepared from the unicellular cyanophyte, Anacystis nidulans, by enzymatic digestion of the cell wall with 0.1% lysozyme. The yield of protoplasts from intact algal cells was approx. 50%. Incorporation of L-[U-14C]leucine into cold trichloroacetic acid-insoluble material from protoplasts preparations was linear for 1.5 h and continued for an additional 2.5 h. Incorporation of radiolabeled leucine into hot trichloroacetic acid-insoluble material from protoplast preparations demonstrated protein synthesis in protoplasts in vitro. Phycocyanin is the principal phycobiliprotein and allophycocyanin is a minor phycobiliprotein in A. nidulans cells. The light-absorbing chromophore of both of these phycobiliproteins is the linear tetrapyrrole (bile pigment), phycocyanobilin. Radiolabeled phycocyanin and allophycocyanin were isolated from protoplast preparations which had been incubated with L-[U-14]leucine or delta-amino[4-14C] levulinic acid (a precursor of phycocyanobilin). The radio-labeled phycobiliproteins were purified by ammonium sulfate fractionation and ion-exchange chromatography on brushite columns. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled leucine) was 106 000 and 82 000 dpm/mg, respectively. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled delta-aminolevulinic acid) was 33 000 and 38 000 dpm/mg, respectively. Phycobiliproteins from protoplasts incubated with radiolabeled leucine were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 25% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated ratioactivity in phycocyanin and allophycocyanin (in brushite column eluates) comigrated with the subunits of these phycobiliproteins on sodium dodecyl sulfate-polyacrylamide gels. Chromic acid degradation of phycobiliproteins from protoplast preparations incubated with delta-amino[4-14C] levulinic acid yielded radiolabeled imides which were derived from the phycocyanobilin chromophore. Imides from radiolabeled phycobiliproteins isolated from protoplast preparations incubated with L-[U-14C]leucine did not contain radioactivity. These results show that both the apoprotein and tetrapyrrolic moieties of phycocyanin and allophycocyanin were synthesized in A. nidulans protoplasts in vitro.

Incorporation of L-azetidine-2-carboxylic acid into hemoglobin S in sickle erythrocytes in vitro.

L-Azetidine-2-carboxylic acid, the naturally occurring lower homologue of L-proline, is incorporated into hemoglobin S (sickle hemoglobin) in vitro. Sickle erythrocytes from patients with sickle cell anemia incubated with L-[3H] azetidine-2-carboxylate synthesized radiolabeled hemoglobin which when isolated from cell lysates, co-chromatographed with hemoglobin S on DEAE-cellulose columns. The alpha/beta ratio of azetidine carboxylate incorporation into the globin chains of sickle hemoglobin was 0.94, which is consistent with the presence of four proline residues in each polypeptide chain. Incorporation of azetidine carboxylate into hot trichloroacetic acid-insoluble material in sickle erythrocytes indicated that the homologue was present in the polypeptide backbone of the globin chains of sickle hemoglobin. Amino acid analysis of the hot trichloroacetic acid-insoluble material from sickle erythrocytes which had been incubated with radiolabeled azetidine carboxylate indicated that 75% of the radioactivity could be accounted for as intact homologue while 20% of the radioactivity co-chromatographed with alanine. These results suggest that azetidine carboxylate is incorporated unaltered into hemoglobin S in addition to being metabolized to alanine in sickle erythrocytes prior to incorporation into protein. The kinetics of thermal precipitation of hemoglobin S (oxygen ligand) into which radioactive azetidine carboxylate or radioactive proline had been incorporated in vitro is identical. This observation, together with the behavior of hemoglobin S and the globin chains from hemoglobin S containing azetidine carboxylate during ion-exchange chromatography, indicates that homologue replacement of prolyl residues does not significantly alter the overall charge or stability of the hemoglobin S tetramer. Azetidine carboxylate did not inhibit uptake of radiolabeled proline by sickle erythrocytes suggesting that the homologue does not adversely affect amino acid transport in these cells.

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.

The amino acid sequence of human beta-microseminoprotein.

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.

Structural characterization of the isoenzymatic forms of human myeloperoxidase.

Myeloperoxidase from human neutrophils was isolated by ion-exchange and gel-filtration chromatography and shown by SDS-polyacrylamide gel electrophoresis to be comprised of alpha and beta subunits with apparent Mr values of 58,000 and 15,000, respectively. The apparent Mr of the native protein was 130,000-140,000, indicating that the holoenzyme has the quaternary structure alpha 2 beta 2. Automated Edman degradation of the separated alpha and beta subunits showed that the amino-terminal sequences were different from one another and demonstrated no sequence microheterogeneity. Comparison of these sequences with those in the National Biomedical Research Foundation data bank of protein sequences revealed that the subunits of human myeloperoxidase were not homologous to any known protein. Myeloperoxidase purified from HL-60 cells grown in culture demonstrated the same alpha 2 beta 2 subunit structure. Three isoenzymes of myeloperoxidase, prepared by gradient elution from a CM-Sepharose column, underwent quantitative analysis. No structural basis for the different elution pattern of the myeloperoxidase isoenzymes was discerned by amino-acid analysis, N-terminal sequence, polyacrylamide gel electrophoresis, or digestion with neuraminidase or enzymes known to cleave N-linked heterosaccharides. The structural basis for the myeloperoxidase isoenzymes of human neutrophils, each possessing equivalent activity, is not apparent from these studies.

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli.

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a "sparing effect" on proline in the mutant. The incorporation of L-[U-14C]proline and L-[3H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologue was incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.

Proteins in whole saliva during the first year of infancy.

During the first year of an infant's life, the oral environment is subject to drastic changes that coincide with the eruption of teeth. Proteins in saliva are important for protecting oral surfaces and provide receptors for bacterial adhesins. The objective of this longitudinal study was to monitor the general composition and expression of proteins in whole saliva of infants, to prove the hypothesis that expression of certain proteins changes during infant development, and might be associated with tooth eruption. The results showed a remarkable constancy in the overall pattern of salivary proteins and glycoproteins during infancy. Exceptions were the mucins and albumin. The mucins are expressed differentially, with first MUC7 and later MUC5B being predominant. Albumin, a marker of serum leakage, started to rise in whole saliva preceding tooth eruption. Thus, the expression of only few proteins appears to be changed during infant development.

Human serum alpha 2HS-glycoprotein modulates in vitro bone resorption.

We previously isolated a family of bone-resorbing proteins from human cancer ascites fluid and established that the three purified bone-resorbing proteins were chemically and immunochemically related to each other and to alpha-2HS glycoprotein (alpha 2HS). After this finding we purified the normal human serum counterpart of these ascites proteins and studied its effects on bone resorption. The bone-resorbing properties of normal human serum alpha 2HS were examined in vitro over a wide dose range. This normal human serum glycoprotein had a biphasic effect on 45Ca2+ release from bone. More specifically, this protein stimulated bone resorption at the lower concentrations tested, with a maximum effect [treated over control ratio of 2.5 +/- 0.30 (+/- SE); P less than 0.01] at 40 micrograms/mL. In contrast, at doses above 40 micrograms/mL, a sharp decline in calcium mobilization occurred, with a return to baseline occurring above 80 micrograms/mL. These results suggest that serum alpha 2HS may participate in the regulation of bone metabolism in vivo.

Candidacidal activity of human salivary histatin recombinant variants produced by site-directed mutagenesis.

Histatin 5 (Hst5) is a 24-amino acid (aa) member of the Hst family that is found in human salivary secretions and exhibits candidacidal activity. Hst5 contains a 13-aa region that alone is capable of killing fungal pathogens and is referred to as the functional domain. To investigate the role of specific aa located within the functional domain, the pRSET bacterial expression system was used to produce recombinant Hst5 (re-Hst5) and several re-variants that were generated by site-directed mutagenesis. The vector pRSETC expresses genes of interest as fusion proteins attached to the carboxy end of an N-terminal His6 tag that binds to nickel (Ni2+). The re-variants were generated using the polymerase chain reaction (PCR) and had Gly substituted for either the His, Glu or Lys/Arg within the functional domain. PCR products that encoded either the wild-type or variant forms of re-Hst5 were inserted into pRSETC and produced as fusion proteins which were affinity purified from cell lysates by Ni(2+)-Sepharose chromatography. Fusion proteins were digested with CNBr and re-Hsts were purified by reversed-phase high performance liquid chromatography (RP-HPLC). Re-Hsts were tested in bioassays to measure the ability to kill both Candida albicans (C. albicans) blastoconidia and spheroplasts which were generated by removal of the cell wall. In both assays, re-Hst5 displayed dose-dependent candidacidal activity that was nearly identical to that of native Hst5 purified from human salivary secretions. Re-Hst5 variants with either Glu or Lys/Arg substitutions demonstrated significantly lower candidacidal activity in both assays, while the variant with His mutated showed essentially no activity at physiological concentrations. These results indicate that acidic and basic aa within the functional domain contribute to candidacidal activity and that the His are essential for candidacidal activity. Additionally, since C. albicans spheroplasts were also susceptible to Hsts, the cell wall is not an essential component in the Hst mechanism of candidacidal action.

Recombinant histatins: functional domain duplication enhances candidacidal activity.

Histatin 3 (Hst3) is a 32-amino-acid (aa) His-rich protein with antimicrobial activity found in human salivary secretions. To explore further the structure/function relationship of Hst, we utilized a bacterial system for the efficient production of recombinant Hst3 (re-Hst3) and Hst variants. Previously, we demonstrated that the middle portion of Hst3 (aa 13-24) contains the functional domain responsible for killing Candida albicans. Using PCR and splice overlap extension, a Hst variant (re-Hst3rep) was made in which the functional domain was repeated in tandem. Using the pRSET bacterial expression system, re-Hst3 and the variant re-Hst3rep were produced as chimeric fusions and were isolated from bacterial sonicates by affinity chromatography. Affinity purified fusion proteins were digested with CNBr and re-Hst were separated from their fusion partners by reverse-phase high-performance liquid chromatography. The activity of re-Hst3 and re-Hst3rep was compared to that of native Hst3 from human salivary secretions in the C. albicans killing assay. The LD50 values for candidacidal activity of native Hst3, re-Hst3 and re-Hst3rep were 7.2, 6.8 and 4.1 nmol/ml, respectively. At lower concentrations re-Hst3rep was five times more active than native Hst3 or re-Hst3 and at even lower concentrations re-Hst3rep exhibited significant candidacidal activity while native Hst3 and re-Hst3 were inactive. These results demonstrate an expression system for production of biologically active functional Hst and Hst variants and shows that repetition of the functional domain of Hst3 enhances candidacidal activity.

Zinc and copper bind to unique sites of histatin 5.

Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5, a human salivary protein. Proton nuclear magnetic resonance (NMR) spectra were obtained to investigate the specificity of metal binding to the seven histidyl, one aspartyl and one glutamyl amino acid side-chains of histatin 5 in aqueous solutions. Three C(epsilon1)-H histidyl and the C(gamma)-H glutamyl resonances of histatin 5 were selectively altered in spectra of solutions containing three equivalents of zinc. Copper binding to histatin 5 resulted in a reduced intensity of C(beta)-H aspartyl resonances, while no evidence for calcium binding was found. These results indicate that zinc binding to histatin 5 involves His-15 present within the -H-E-X-X-H- zinc binding motif, and copper binding occurs within the N-terminal D-S-H-, ATCUN motif.

Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain.

Histatin 5 is a low molecular weight salivary protein which is known to exhibit inhibitory activity against several proteinases, including the cysteine proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine proteinase clostripain derived from the pathogen Clostridium histolyticum. Using a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to that of leupeptin, a known competitive inhibitor of clostripain. It was found that the concentration of histatin 5 required to inhibit 50% of clostripain activity was 23.6+/-1.6 nM. Kinetic analysis revealed that histatin 5 is a competitive inhibitor of clostripain with an inhibition constant (K(i)) of 10 nM. The K(i) for the inhibition of clostripain activity against nitroanilide substrate by leupeptin was found to be 60 nM, significantly higher than that of histatin 5. Thus, histatin 5 inhibits clostripain more effectively than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incubated at physiologic concentrations with clostripain. The potent inhibition of clostripain by histatin 5 points towards the possibility that this protein may prevent establishment of clostridial infections and therefore may have significant potential for the treatment of diseases associated with this enzyme.

The association of elevated plasma cortisol and early atherosclerosis as demonstrated by coronary angiography.

To study the association of plasma cortisol and coronary atherosclerosis, we elected 71 male outpatients who had coronary angiography as part of their evaluation at our facility. Forty-eight percent of the angiograms showed no evidence of coronary artery disease (CAD), 20% showed mild CAD, and 32% showed moderate to severe CAD. We found significant correlations between elevated serial morning plasma cortisols and moderate to severe coronary atherosclerosis. Using the odds ratio, we compared plasma cortisol to the major risk factors for coronary artery disease. Plasma cortisol was second only to serum cholesterol as a discriminator in our patient population between diseased and non-diseased patients. We found a significant correlation between plasma cortisol and cholesterol, blood pressure, and smoking- the three cardinal risk factors for CAD. The highest degree of correlation was found between cortisol and cholesterol. The possible significance of the association of cortisol and the major risk factors for CAD is discussed.

Cortisol and the hypercholesterolemia of pregnancy and labor.

Lipids and lipoproteins are known to increase substantially during pregnancy and to decrease rapidly after delivery. The factors responsible for the changes have not been identified, however, they could be related to changes in one or more of the endocrine hormones. Since studies relating lipid and lipoprotein changes to cortisol or estradiol concentrations have not been made, we sought to perform such a study. For this study, we measured cholesterol, HDL-C, VLDL/LDL-C, cortisol, and estradiol concentrations from early gestation through delivery in 32 normal pregnant women. During the course of pregnancy, cholesterol increased from 145 to 211 mg/dl (45%); plasma cortisol increased from 8.6 to 17.8 micrograms/dl (107%); and urinary cortisol increased from 0.10 to 0.177 microgram/mg of creatinine (72%). Further significant increases in cholesterol (256 mg/dl, P less than 0.005) and cortisol (77.6 micrograms/dl, P less than 0.001) occurred during labor, and both decreased after delivery. Pooled correlations were calculated and both cholesterol and VLDL/LDL-cholesterol concentrations were found to be related to plasma cortisol as well as to urinary cortisol (P less than 0.001). Plasma estradiol concentrations increased during pregnancy, but not during labor. The results suggest that the increases in cholesterol during pregnancy and labor could be due, in part, to the metabolic and stress-related increases in cortisol. The studies also suggest that both pregnancy and labor and delivery might be useful "natural" models for studying hormonal mechanisms involved in lipid and lipoprotein metabolism.

Relation between high density lipoprotein cholesterol and coronary artery disease in asymptomatic men.

The well established inverse relation of high density lipoprotein cholesterol (HDL) and the risk of coronary artery disease was tested in a cross-sectional group of 572 asymptomatic aircrew members who were being screened for risk of coronary artery disease. A battery of tests was performed, including determinations of fasting serum cholesterol, HDL cholesterol and triglycerides and performance of a maximal symptom-limited exercise tolerance test. Of the 572 patients, 132 also had an abnormal S-T segment response to exercise testing or were otherwise believed to have an increased risk of organic heart disease and subsequently underwent coronary angiography. Significant coronary artery disease was found in 16 men and minimal or subcritical coronary disease in 14; coronary angiograms were normal in the remaining 102 men. The remaining 440 men, who were believed to have a 1 percent chance of having coronary artery disease by sequential testing of risk factors and treadmill testing, had a mean cholesterol level of 213 mg/100 ml, a mean HDL cholesterol of 51 mg/100 ml and a mean cholesterol/HDL ratio of 4.4. The mean values of cholesterol, HDL cholesterol and cholesterol/HDL cholesterol did not differ significantly in men with normal angiographic finding and those with subcritical coronary disease. However, 14 of 16 men with coronary artery disease had a cholesterol/HDL ratio of 6.0 or more whereas only 4 men with normal coronary arteries had a ratio of 6.0 or more. Of the classical coronary risk factors evaluated, the cholesterol/HDL ratio of 6.0 or more had the highest odds ratio (172:1). It appears that determination of HDL cholesterol level helps to identify asymptomatic persons with a greater risk of having coronary artery disease.

Interstitial deletion involving most of Yq.

Males with a Yq deletion are well described, but few have been studied with both cytogenetic and molecular techniques to define the deletion and relate it to the phenotype. This study reports an analysis of cells obtained from a college student with azoospermia, short stature, and a small penis. Cytogenetic analysis indicated that the entire Yq was deleted, but DNA hybridization showed that a portion of Yq12 remained. We conclude that the deletion is interstitial.