Abnormalities in arterial-ventricular coupling in older healthy persons are attenuated by sodium nitroprusside

The coupling between arterial elastance (E(A); net afterload) and left ventricular elastance (E(LV); pump performance), known as E(A)/E(LV), is a key determinant of cardiovascular performance and shifts during exercise due to a greater increase in E(LV) versus E(A). This normal exercise-induced reduction in E(A)/E(LV) decreases with advancing age. We hypothesized that sodium nitroprusside (SNP) can acutely ameliorate the age-associated deficits in E(A)/E(LV). At rest and during graded exercise to exhaustion, E(A) was characterized as end-systolic pressure/stroke volume and E(LV) as end-systolic pressure/end-systolic volume. Resting E(A)/E(LV) did not differ between old (70 ± 8 yr, n = 15) and young (30 ± 5 yr, n = 17) subjects because of a tandem increase in E(A) and E(LV) in older subjects. During peak exercise, a blunted increase in E(LV) in old (7.8 ± 3.1 mmHg/ml) versus young (11.4 ± 6.5 mmHg/ml) subjects blunted the normal exercise-induced decline in E(A)/E(LV) in old (0.25 ± 0.11) versus young (0.16 ± 0.05) subjects. SNP administration to older subjects lowered resting E(A)/E(LV) by 31% via a reduction in E(A) (10%) and an increase in E(LV) (47%) and lowered peak exercise E(A)/E(LV) (36%) via an increase in E(LV) (68%) without a change in E(A). Importantly, SNP attenuated the age-associated deficits in E(A)/E(LV) and E(LV) during exercise, and at peak exercise E(A)/E(LV) in older subjects on drug administration did not differ from young subjects without drug administration. In conclusion, some age-associated deficiencies in E(A)/E(LV), E(A), and E(LV), in older subjects can be acutely abolished by SNP infusion. This is relevant to common conditions in older subjects associated with a significant impairment of exercise performance such as frailty or heart failure with preserved ejection fraction.     

ATP-binding modes and functionally important interdomain bonds of sarcoplasmic reticulum Ca2+-ATPase revealed by mutation of glycine 438, glutamate 439, and arginine 678

ATP binds to sarcoplasmic reticulum Ca(2+)-ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca(2)E(1)P --> E(2)P and E(2)P --> E(2) reactions) and Ca(2+) binding (E(2) --> Ca(2)E(1)). In some of the Ca(2+)-ATPase crystal structures, Arg(678) and Glu(439) seem to be involved in the binding of nucleotide or an associated Mg(2+) ion. We have replaced Arg(678), Glu(439), and Gly(438) with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP. The results point to the key role of Arg(678) in nucleotide binding and to the importance of interdomain bonds Glu(439)-Ser(186) and Arg(678)-Asp(203) in stabilizing the E(2)P and E(2) intermediates, respectively. Mutation of Arg(678) had conspicuous effects on ATP/MgATP binding to the E(1) form and ADP binding to Ca(2)E(1)P, as well as ATP/MgATP binding in modulatory modes to E(2)P and E(2), whereas the effects on ATP/MgATP acceleration of the Ca(2)E(1)P --> E(2)P transition were small, suggesting that the nucleotide that accelerates Ca(2)E(1)P --> E(2)P binds differently from that modulating the E(2)P --> E(2) and E(2) --> Ca(2)E(1) reactions. Mutation of Glu(439) hardly affected nucleotide binding to E(1), Ca(2)E(1)P, and E(2), but it led to disruption of the modulatory effect of ATP on E(2)P --> E(2) and acceleration of the latter reaction, indicating that ATP normally modulates E(2)P --> E(2) by interfering with the interaction between Glu(439) and Ser(186). Gly(438) seems to be important for this interaction as well as for nucleotide binding, probably because of its role in formation of the helix containing Glu(439) and Thr(441).     

The sex-specific impact of systolic hypertension and systolic blood pressure on arterial-ventricular coupling at rest and during exercise

In healthy subjects the arterial system and the left ventricle (LV) are tightly coupled at rest to optimize cardiac performance. Systolic hypertension (SH) is a major risk factor for heart failure and is associated with structural and functional alterations in the arteries and the LV. The effects of SH and resting systolic blood pressure (SBP) on arterial-ventricular coupling (E(a)I/E(LV)I) at rest, at peak exercise, and during recovery are not well described. We noninvasively characterized E(a)I/E(LV)I as end-systolic volume index/stroke volume index in subjects who were normotensive (NT, n = 203) or had SH (brachial SBP > or =140 mmHg, n = 79). Cardiac volumes were measured at rest and throughout exhaustive upright cycle exercise with gated blood pool scans. E(a)I/E(LV)I reserve was calculated by subtracting peak from resting E(a)I/E(LV)I. At rest, E(a)I/E(LV)I did not differ between SH and NT men but was 23% (P = 0.001) lower in SH vs. NT women. E(a)I/E(LV)I did not differ between SH and NT men or women at peak exercise or during recovery. Nevertheless, E(a)I/E(LV)I reserve was 61% (P < 0.001) lower in SH vs. NT women. Similarly, resting SBP (as a continuous variable) was not associated with E(a)I/E(LV)I in men (beta = -0.12, P = 0.17) but was inversely associated with E(a)I/E(LV)I in women (beta = -0.47, P < 0.001). SH and a higher resting brachial SBP are associated with a lower E(a)I/E(LV)I at rest in women but not in men, and SH women have an attenuated E(a)I/E(LV)I reserve. Whether a smaller E(a)I/E(LV)I reserve leads to functional limitations warrants further examination.     

Fourier transform infrared evidence for early deprotonation of Asp(85) at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).     

Evidence for the formation of Michael adducts from reactions of (E,E)-muconaldehyde with glutathione and other thiols

Glutathione induces the rapid isomerization of (Z,Z)-muconaldehyde to (E,E)-muconaldehyde via (E,Z)-muconaldehyde, probably via reversible Michael addition of the thiol to one of the enal moieties of the muconaldehyde. Reactions of (E,E)-muconaldehyde with glutathione (in the presence and absence of equine glutathione S-transferase), phenylmethanethiol, N-acetyl-l-cysteine, and N-acetyl-l-cysteine methyl ester were investigated using mass spectrometric techniques. In each case, evidence was obtained for the formation of Michael adducts, e.g., reaction between (E,E)-muconaldehyde and glutathione gave 4-glutathionyl-hex-2-enedial and 3,4-bis-glutathionyl-hexanedial. These experiments suggest that (Z,Z)-muconaldehyde, a putative metabolite of benzene, could lead to the long established urinary metabolite of benzene, (E,E)-muconic acid, via glutathione-mediated isomerization to (E,E)-muconaldehyde.     

Single-beat estimation of right ventricular end-systolic pressure-volume relationship

Assessment of right ventricular (RV) contractility from end-systolic pressure-volume relationships (ESPVR) is difficult due to problems in measuring RV instantaneous volume and to effects of changes in RV preload or afterload. We therefore investigated in anesthetized dogs whether RV ESPVR and contractility can be determined without measuring RV volume and without changing RV preload or afterload. The maximal RV pressure of isovolumic beats (P(max)) was predicted from isovolumic portions of RV pressure during ejecting beats and compared with P(max) measured during the first beat after pulmonary artery clamping. In RV pressure-volume loops obtained from RV pressure and integrated pulmonary arterial flow, end-systolic elastance (E(es)) was assessed as the slope of P(max)-derived ESPVR, pulmonary artery effective elastance (E(a)) as the slope of end-diastolic to end-systolic relation, and coupling efficiency as the E(es)-to-E(a) ratio (E(es)/E(a)). Predicted P(max) correlated with observed P(max) (r = 0.98 +/- 0.02). Dobutamine increased E(es) from 1.07 to 2.00 mmHg/ml and E(es)/E(a) from 1.64 to 2.49, and propranolol decreased E(es)/E(a) from 1.64 to 0.91 (all P < 0.05). After adrenergic blockade, preload reduction did not affect E(es), whereas hypoxia and arterial constriction markedly increased E(a) and somewhat increased E(es) due to the Anrep effect. Low preload did not affect E(es)/E(a) and high afterload decreased E(es)/E(a). In conclusion, in the right ventricle 1) P(max) can be calculated from normal beats, 2) P(max) can be used to determine ESPVR without change in load, and 3) P(max)-derived ESPVR can be used to assess ventricular contractility and ventricular-arterial coupling efficiency.     

(4E)-dehydrocitrals [(2E,4E)- and (2Z,4E )-3,7-dimethyl-2,4,6-octatrienals] from acarid mite Histiogaster sp. A096 (Acari: Acaridae)

A mixture of two monoterpenes was obtained as the opisthonotal gland secretion from unidentified Histiogaster sp. A096 (Acari: Acaridae), and their structures were elucidated to be (4E)-dehydrocitrals [(2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienals] by GC/MS, GC/FT-IR, UV and 1H-NMR spectra. Both isomers of (4E)-dehydrocitral prepared by syntheses in 4 steps from 3-methyl-2-butenal with 34.2% yields (based on the ylide) were separated by column chromatography into the (2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienal. Mass spectra together with GC retention times of the purified natural (4E)-dehydrocitrals were identical with those of synthetic (2E,4E)-3,7-dimethyl-2,4,6-octatrienal and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienal. The geometry at the 2-C position of both synthetic (4E)-dehydrocitrals was confirmed by NOESY analyses. This is the first identification of (4E)-dehydrocitrals from the animal kingdom.     

Apolipoprotein E enhances hepatic lipase-mediated hydrolysis of reconstituted high-density lipoprotein phospholipid and triacylglycerol in an isoform-dependent manner

This study compares the kinetics of hepatic lipase (HL)-mediated phospholipid and triacylglycerol hydrolysis in spherical, reconstituted high-density lipoproteins (rHDL) that contain either apolipoprotein E2 (apoE2), apoE3, apoE4, or apoA-I as the sole apolipoprotein. HL-mediated phospholipid hydrolysis was assessed by incubating various concentrations of rHDL that contained only cholesteryl esters (CE) in their core, (E2/CE)rHDL, (E3/CE)rHDL, (E4/CE)rHDL, and (A-I/CE)rHDL, with a constant amount of HL. The rate of phospholipid hydrolysis was determined as the formation of nonesterified fatty acid mass. HL-mediated triacylglycerol hydrolysis was assessed in rHDL containing CE, unlabeled triacylglycerol, and [(3)H]triacylglycerol in their core, (E2/TG)rHDL, (E3/TG)rHDL, (E4/TG)rHDL, and (A-I/TG)rHDL. Triacylglycerol hydrolysis was determined as the ratio of (3)H-labeled hydrolysis products to (3)H-labeled unhydrolyzed triacylglycerol. The rates of phospholipid hydrolysis in the (E2/CE)rHDL, (E3/CE)rHDL, and (E4/CE)rHDL were significantly greater than that in the (A-I/CE)rHDL. The rates of triacylglycerol hydrolysis were also greater in the (E2/TG)rHDL, (E3/TG)rHDL, and (E4/TG)rHDL compared to the (A-I/TG)rHDL, although to a lesser degree than observed with phospholipid hydrolysis. Furthermore, the rates of both phospholipid and triacylglycerol hydrolyses were greater in the (E2)rHDL than in either the (E3)rHDL or the (E4)rHDL. These results show that apoE increases the rate of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL and that this influence is isoform dependent.     

Coccidia (Apicomplexa: Eimeriidae) from sciurid rodents (Eutamias, Sciurus, Tamiasciurus spp.) from the western United States and northern Mexico with description of two new species

Since May 1979, 190 rodents in the family Sciuridae, representing three genera and nine species, have been collected in the western United States and northern Mexico and examined for coccidia; 71 (37%) had coccidian oocysts in their feces. These included 2 of 12 (17%) Eutamias canipes; 7 of 12 (58%) E. dorsalis; 18 of 50 (36%) E. merriami; 33 of 96 (34%) E. obscurus; 3 of 4 (75%) E. townsendii; 3 of 9 (33%) Sciurus aberti; 1 of 1 S. griseus; 1 of 1 Tamiasciurus hudsonicus mogollonensis; and 3 of 5 (60%) T. mearnsi. The following coccidians were identified from infected rodents: Eimeria cochisensis n. sp. and Eimeria dorsalis n. sp. from E. canipes, E. cochisensis, E. dorsalis, and E. tamiasciuri from E. dorsalis, E. dorsalis and E. tamiasciuri from E. merriami; E. cochisensis, E. dorsalis, E. tamiasciuri, and E. wisconsinensis from E. obscurus; E. cochisensis and E. dorsalis from E. townsendii; E. ontarioensis and E. tamiasciuri from S. aberti; E. tamiasciuri from S. griseus; E. tamiasciuri and E. toddi from T. h. mogollonensis; and E. tamiasciuri from T. mearnsi. Sporulated oocysts of Eimeria dorsalis n. sp. were ovoid, 21.9 x 16.8 (17-24 x 14-20) micrometer with sporocysts ovoid, 11.5 x 6.9 (10-14 x 6-8) micrometer. Sporulated oocysts of Eimeria cochisensis n. sp. were spheroid to subspheroid, 16.7 x 15.3 (15-18 x 14-17) micrometer, with sporocysts ovoid, 8.4 x 5.6 (6-11 x 4-7) micrometer. Fifty-five of 71 (77%) infected hosts had oocysts of only one eimerian species in their feces at the time they were examined. One eimerian, E. tamiasciuri, was found in seven of nine host species in three genera. A list is provided of all eimerians (22, including the species described here) that have been described in the literature from Eutamias, Sciurus, and Tamiasciurus spp.     

Dietary intake of artificial food color additives containing food products by school-going children

Nutritional risk in children is associated with food safety. This is the first study to identify the food type consumed by 6–17-year-old school-going children in Saudi Arabia. Eight permitted artificial food color additives, including Tartrazine (E102), Sunset Yellow (E110), Carmoisine (E122), Allura Red (E129), Indigo Carmine (E132), Brilliant Blue (E133), Fast Green (E143), and Black PN (E151), and two non-permitted ones, Erythrosine (E127) and Red 2G (E128), were determined using 24-h dietary recall questionnaires. Artificial color additives in 839 food products were divided into nine categories, including biscuits, cakes, chocolates, chips, ice cream, juices and drinks, candy, jelly, and chewing gum, are determined using high performance liquid chromatography and diode array detector. The results indicated a high intake of juices and drinks, ice cream, and cakes, but low consumption of chewing gum among school-going children. Among the permitted artificial food color additives, Brilliant Blue (E133) (54.1%) and Tartrazine (E102) (42.3%) were the most commonly used. Sunset Yellow (E110) in one chocolate sample, Tartrazine (E102) and Sunset Yellow (E110) in one and two juice and drink samples, respectively, and Brilliant Blue (E133) in two candy samples exceeded the permitted level. Therefore, further investigations are needed to provide insights into the possible adverse health effects of high intake of these additives in artificial food coloring on the test population are warranted.     

Structural bases for the specific interactions between the E2 and E3 components of the Thermus thermophilus 2-oxo acid dehydrogenase complexes

Pyruvate dehydrogenase (PDH), branched-chain 2-oxo acid dehydrogenase (BCDH) and 2-oxoglutarate dehydrogenase (OGDH) are multienzyme complexes that play crucial roles in several common metabolic pathways. These enzymes belong to a family of 2-oxo acid dehydrogenase complexes that contain multiple copies of three different components (E1, E2 and E3). For the Thermus thermophilus enzymes, depending on its substrate specificity (pyruvate, branched-chain 2-oxo acid or 2-oxoglutarate), each complex has distinctive E1 (E1p, E1b or E1o) and E2 (E2p, E2b or E2o) components and one of the two possible E3 components (E3b and E3o). (The suffixes, p, b and o identify their respective enzymes, PDH, BCDH and OGDH.) Our biochemical characterization demonstrates that only three specific E3*E2 complexes can form (E3b*E2p, E3b*E2b and E3o*E2o). X-ray analyses of complexes formed between the E3 components and the peripheral subunit-binding domains (PSBDs), derived from the corresponding E2-binding partners, reveal that E3b interacts with E2p and E2b in essentially the same manner as observed for Geobacillus stearothermophilus E3*E2p, whereas E3o interacts with E2o in a novel fashion. The buried intermolecular surfaces of the E3b*PSBDp/b and E3o*PSBDo complexes differ in size, shape and charge distribution and thus, these differences presumably confer the binding specificities for the complexes.     

A nongenomic action of 17beta-estradiol as the mechanism underlying the acute suppression of secretion of luteinizing hormone

The objective of the present study was to determine the ability of 17beta-estradiol (E(2)) and conjugated forms of E(2) (E(2) conjugated to BSA [E(2)-BSA] and a novel conjugate, E(2) conjugated to a small peptide [E(2)-PEP]) to prevent the GnRH-induced secretion of LH and to determine the role of estradiol receptors (ERs) and ER subtypes (ERalpha, also known as ESR1, and ERbeta, also known as ESR2) in the mediation of the acute action of E(2) in primary cultures of ovine pituitary cells. Preincubation of cells for 15 min with E(2), E(2)-BSA, or E(2)-PEP prevented the GnRH-induced secretion of LH (P < 0.01). Treatment of cells with nonestrogenic steroid hormones did not affect secretion of LH when given alone, nor did these steroids impair the E(2)-induced inhibition of LH secretion (P > 0.1). Likewise, treatment of cells with the ER-antagonists tamoxifen, hydroxytamoxifen, or ICI 182 780 did not affect (P > 0.1) secretion of LH when given alone but did prevent (P < 0.01) the inhibition by E(2) and the E(2)-conjugates on GnRH-induced secretion of LH. When cells were treated with subtype-selective ER agonists, the ERalpha agonist (propylpyrazole-triol), but not the ERbeta agonist (diarylpropionitrile), decreased (P < 0.01) the GnRH-induced secretion of LH. In conclusion, the rapidity by which E(2) prevented GnRH-induced release of LH in ovine pituitary cells suggests that this inhibition is mediated via a nongenomic action of E(2). The inhibition of GnRH-induced secretion of LH proved to be steroid specific and mediated by ERs. It may occur specifically through ERalpha. The fact that E(2)-BSA or E(2)-PEP mimicked the action of E(2) suggests that this effect was mediated by an ER associated with the plasma membrane.     

New taxa, new records and redefined concepts in the Elaphoglossum sect. Elaphoglossum subsec. Pachyglossa (Lomariopsidaceae) from Mexico and Central America

Twelve new species are described in the taxonomically difficult Elaphoglossum (sect. Elaphoglossum): Elaphoglossum angustifrons A. Rojas, E. delgadilloanum A. Rojas, E. ellipticifolium A. Rojas, E. incognitum A. Rojas, E. mesoamericanum A. Rojas, E. nicaraguese A. Rojas, E. polypodium A. Rojas, E. tejeroanum A. Rojas, E. reptans A. Rojas, E. terrestre A. Rojas, E. variabile A. Rojas and E. zavale A. Rojas. Also, E. latifolium (Sw.) J. Sm., E. sartorii (Liebm.) Mickel and E. viride (E. Fourn.) C. Chr. are amended, E. andicola (Fée) T. Moore and E. sporadolepis (Kunze ex Kuhn) T. Moore are reported.     

蓍草化学成分研究

研究蓍草Achillea alpine L.全草的化学成分。采用大孔树脂、ODS、Sephadex LH-20凝胶柱色谱和pre-HPLC等方法分离与纯化,运用NMR、MS等波谱技术鉴定化合物结构。从蓍草95%乙醇提取物中分离得到16个化合物,分别是(2 E,4 E)-N-(2-methylbutyl)deca-2,4-dienamide(1)、墙草碱(2)、(E,E,Z)-2,4,8-decatrienoicacid isobutylamide-8,9-dehydropellitorine(3)、N-2′-methylbutyl-(E,E)-2,4-decadienam(4)、methyl-(E,E)-2,4,9-oxooctadeca-10,12-dienoate(5)、(S)-14-(E,E)-10,12-methyl 14-hydroxy-9-oxo-octadeca-10,12-dienoate(6)、(E,E)-2,4-undecadiene-8,10-diynamide-N-(2-methylpropyl)(7)、(E,E)-2,4-decadienoic acid p-hydroxyphenethylamide(8)、sinapyl alcohol diisovalerate(9)、(S)-13-hydroxyoctadeca-(Z,E)-9,11-dienoic acid(10)、(E,E)-2,4-decadienamide acid p-methoxyphenethylamide(11)、erythro-N-isobutyl-4,5-dihydroxy-2-(E)-decenamide(12)、3-O-阿魏酰-奎宁酸(13)、肉桂酸(14)、绿原酸(15)、3-O-咖啡酰-5-O-阿魏酰奎宁酸(16)。化合物1是一个新的酰胺类化合物;化合物4~6、9、10、12、13、16为首次从该属植物中分离得到;化合物8、11为首次从蓍草中分离得到。化合物1~11在四种不同的胃癌细胞株上进行细胞毒活性筛选,结果显示化合物2、5与9在50μM时对MGC-803细胞株具有较弱抑制活性,其抑制率依次为38.7%、34.7%、31.5%。     

des-Tyr1-gamma-endorphin and haloperidol increase pineal gland melatonin levels in rats

The effect of subcutaneously injected DT gamma E (beta-endorphin, (beta E)2-17) on the pineal melatonin level was compared with that of closely related peptides and the neuroleptic drug haloperidol. As found previously, DT gamma E (3 ng/rat and 300 ng/rat) increased the melatonin levels. Similar doses of DT alpha E (beta E 2-16), DT beta E (beta E 2-31), gamma E (beta E 1-17), alpha E (beta E 1-16) and beta E failed to significantly change the melatonin levels in both the dark and the light phase. Haloperidol in a dose of 300 ng/rat exhibited a similar effect as DT gamma E.     

Genetic heterogeneity in familial dysbetalipoproteinemia. The E2(lys146----gln) variant results in a dominant mode of inheritance

As determined by isoelectric focusing, most patients with familial dysbetalipoproteinemia (FD) exhibit the homozygous apolipoprotein (apo) E2E2 phenotype. Only rarely does FD develop in the more common heterozygous phenotypes E3E2 or E4E2. In fact, only 1 to 4% of the E2E2 homozygotes will develop FD. We wondered whether this reduced penetrance of FD in E2E2 homozygotes could be due to additional heterogeneity in the APOE*2 allele. In the literature a number of different mutations causing an E2 isoelectric focusing variant have been described. To study the genetic heterogeneity of the APOE gene, hybridization of enzymatically amplified genomic DNA with mutation-specific oligonucleotide probes was applied. All FD patients (n = 40) with the E2E2 phenotype appeared to be homozygous for the common E2(arg158----cys) mutation. However, all three unrelated patients with the E3E2 phenotype exhibited the rare E2(lys146----gln) mutation due to an A----C substitution at nucleotide position 3,847 of the APOE gene. This mutation was not found among normolipidemic individuals with the E2E2 (n = 13) or E3E2 phenotype (n = 120) selected from a random population sample. Family studies of the three probands heterozygous for the E*2(lys146----gln) allele showed that this rare allele predisposes to FD with high penetrance. We conclude that FD is a genetically heterogeneous disease entity, displaying a recessive mode of inheritance with strongly reduced penetrance in case of the common E2(arg158----cys) variant and with a dominant mode of inheritance with high penetrance in case of the rare E2(lys146----gln) mutant. It should be noted that in this dominant form presymptomatic diagnosis is possible.     

The resolution and characterization of putative ubiquitin carrier protein isozymes from rabbit reticulocytes

The covalent ligation of the 8.6-kDa polypeptide ubiquitin to various cellular target proteins is believed to represent a fundamental regulatory process. In this mechanism, the ATP-coupled activation and subsequent ligation of ubiquitin are catalyzed by separate enzymes (E1 and E3, respectively) functionally linked by ubiquitin carrier protein (E2). Carrier protein has been proposed to constitute a family of isozymes having molecular masses of 14, 17, 20, 24, and 32 kDa whose role is to shuttle activated polypeptide in the form of a high-energy thiol ester intermediate to the carboxyl terminus of ubiquitin. Using a combination of covalent affinity and high performance liquid chromatographic methods, the pututive E2 isozymes have been purified to apparent homogeneity. The E2(14kDa) isozyme resolved into two forms differing in net charge at pH 7.5. All of the E2 isozymes contained only one thiol ester site except for E2(17kDa) and E2(20kDa) which were capable of forming two such adducts per subunit. Thiol ester formation was rapid for the E2 isozymes and required the presence of activating enzyme. In contrast, the reverse reaction of thiol ester transfer from E2 to E1 was kinetically significant for only E2(14kDa), E2(20kDa), and E2(24kDa). The stability of E2(17kDa) and E2(32kDa) to such trapping may reflect a marked shift in binding affinity to E1 upon thiol ester formation. In addition, differential rates for thiol ester formation to each subunit of dimeric E2(14kDa) was also noted. The E2(14kDa) isoforms were approximately 10-fold more active in E3-dependent ubiquitin-protein ligation than either E2(20kDa) or E2(32kDa). Neither E2(17kDa) nor E2(24kDa) supported this reaction. In addition, the thiol ester formed to E2(14kDa) was inherently more reactive since its second order rate constant for the E3-independent transfer of ubiquitin to the small molecular weight nucleophile dithiothreitol was an order of magnitude greater than found for the other isozymes. If these proteins constitute a family of isozymes, they exhibit considerable catalytic diversity.     

Human papillomavirus type 16 E1circumflexE4 contributes to multiple facets of the papillomavirus life cycle

The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation program of the host's stratified epithelia that it infects. E1(circumflex)E4 is a viral protein that has been ascribed multiple biochemical properties of potential biological relevance to the viral life cycle. To identify the role(s) of the viral E1(circumflex)E4 protein in the HPV life cycle, we characterized the properties of HPV type 16 (HPV16) genomes harboring mutations in the E4 gene in NIKS cells, a spontaneously immortalized keratinocyte cell line that when grown in organotypic raft cultures supports the HPV life cycle. We learned that E1(circumflex)E4 contributes to the replication of the viral plasmid genome as a nuclear plasmid in basal cells, in which we also found E1(circumflex)E4 protein to be expressed at low levels. In the suprabasal compartment of organotypic raft cultures harboring E1(circumflex)E4 mutant HPV16 genomes there were alterations in the frequency of suprabasal cells supporting DNA synthesis, the levels of viral DNA amplification, and the degree to which the virus perturbs differentiation. Interestingly, the comparison of the phenotypes of various mutations in E4 indicated that the E1(circumflex)E4 protein-encoding requirements for these various processes differed. These data support the hypothesis that E1(circumflex)E4 is a multifunctional protein and that the different properties of E1(circumflex)E4 contribute to different processes in both the early and late stages of the virus life cycle.     

Apolipoprotein E polymorphism in Saudis

Apolipoprotein E (apoE) genotypes were determined in 165 Saudis. The prevalence of genotype, E3/E3, E3/E4 and E4/E4 was found to be 71, 27 and 2% respectively. The E3/E3 was the most prevalent genotype among the Saudis followed by E3/E4. However, other genotypes E2/E2, E2/E3 and E2/E4 were absent showing the absence of E2 allele in the test population. The high frequencies of the E3 allele (0.845) and E3/E3 genotype (0.71) and absence of E2 allele in Saudis under study are similar to those reported earlier for Native Americans, Mexican-Americans, Mayans, Cayapa, Mazatecan Indians and Mexican Mestizos populations.     

Apolipoprotein E polymorphism in Saudis

Apolipoprotein E (apoE) genotypes were determined in 165 Saudis. The prevalence of genotype, E3/E3, E3/E4 and E4/E4 was found to be 71, 27 and 2% respectively. The E3/E3 was the most prevalent genotype among the Saudis followed by E3/E4. However, other genotypes E2/E2, E2/E3 and E2/E4 were absent showing the absence of E2 allele in the test population. The high frequencies of the E3 allele (0.845) and E3/E3 genotype (0.71) and absence of E2 allele in Saudis under study are similar to those reported earlier for Native Americans, Mexican-Americans, Mayans, Cayapa, Mazatecan Indians and Mexican Mestizos populations.