Alpha A2 Research Articles

First observation of hemoglobin G-Norfolk in the Turkish population

Abstract Objectives Hemoglobinopathies are inherited transition blood diseases associated with globin chains of the hemoglobin. However many mutations have been defined, there may be many of them not defined yet. We here report the first case of those mutations, named Hb G-Norfolk in Turkey. Case presentation A 15 years-old male patient with erythrocytosis was referred to our laboratory for the evaluation of hemoglobinopathy. In chromatographic analysis, an unidentified peak was observed. A similar identification for variant Hb could not be obtained from High-Performance Liquid Chromatography (HPLC) analyzer’s data library. No definitive diagnosis could also be made by different analyzer. Family screening and molecular genetic DNA sequence analysis were carried out. Conclusions Although there were not found any beta gene mutation of neither the patient nor his family, analyses of alpha genes A1 and A2 were performed and abnormal hemoglobines were detected for all of them. This change in the HbA2 gene was at codon85 GAC>AAC (Asp>Asn) in the heterozygous state, known as Hb G-Norfolk [HbA2:c256G>A p.Asp85Asn] based on HbVar database. Abnormal Hb bands detected by HPLC with clinical findings such as erythrocytosis or cyanosis should be investigated by sequence analysis to corroborate alpha and/or beta-globin gene mutations for avoiding misdiagnosis and misinterpretation.

Changes in metabolite profiles caused by genetically determined obesity in mice.

The Berlin Fat Mouse Inbred (BFMI) line harbors a major recessive gene defect on chromosome 3 (jobes1) leading to juvenile obesity and metabolic syndrome. The present study aimed at the identification of metabolites that might be linked to recessively acting genes in the obesity locus. Firstly, serum metabolites were analyzed between obese BFMI and lean B6 and BFMI × B6 F1 mice to identify metabolites that are different. In a second step, a metabolite–protein network analysis was performed linking metabolites typical for BFMI mice with genes of the jobes1 region. The levels of 22 diacyl-phosphatidylcholines (PC aa), two lyso-PC and three carnitines were found to be significantly lower in obese mice compared with lean mice, while serine, glycine, arginine and hydroxysphingomyelin were higher for the same comparison. The network analysis identified PC aa C42:1 as functionally linked with the genes Ccna2 and Trpc3 via the enzymes choline kinase alpha and phospholipase A2 group 1B (PLA2G1B), respectively. Gene expression analysis revealed elevated Ccna2 expression in adipose tissue of BFMI mice. Furthermore, unique mutations were found in the Ccna2 promoter of BFMI mice which are located in binding sites for transcription factors or micro RNAs and could cause differential Ccna2 mRNA levels between BFMI and B6 mice. Increased expression of Ccna2 was consistent with higher mitotic activity of adipose tissue in BFMI mice. Therefore, we suggest a higher demand for PC necessary for adipose tissue growth and remodeling. This study highlights the relationship between metabolite profiles and the underlying genetics of obesity in the BFMI line.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-013-0590-1) contains supplementary material, which is available to authorized users.

Comparison of the effect of simvastatin versus simvastatin/ezetimibe versus rosuvastatin on markers of inflammation and oxidative stress in subjects with hypercholesterolemia

ObjectivesStatins may exhibit anti-inflammatory and antioxidant effects. Whether different statins at equivalent doses or the combination of low-dose statin with ezetimibe have comparable anti-inflammatory and antioxidant effects is unknown. The aim of this study was to compare the effects of simvastatin, simvastatin/ezetimibe or rosuvastatin at equivalent low-density lipoprotein cholesterol lowering doses on inflammation and oxidative stress indices in subjects with hypercholesterolemia. MethodsThis was a pre-specified analysis of a prospective, randomized, open-label, blinded endpoint (PROBE) study. We enrolled one hundred and fifty three (n = 153) hypercholesterolemic subjects who were randomized to receive simvastatin 40 mg or simvastatin/ezetimibe 10/10 mg or rosuvastatin 10 mg daily. Plasma 8-Epi prostaglandin F2 alpha (8-epiPGF2a), oxidized LDL (oxLDL) and lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and mass were measured at baseline and following 12 weeks of treatment. ResultsA significant reduction in plasma 8-isoprostane and oxLDL levels was observed in all treatment groups [by 10%, 8% and 6% (p < 0.05 compared with baseline) and 41%, 40% and 39% (p < 0.001 compared with baseline) in simvastatin, simvastatin/ezetimibe and rosuvastatin groups, respectively]. In all treatment groups a significant reduction in total plasma Lp-PLA2 activity and mass was observed (by 36%, 31% and 38% and 36%, 32% and 32% for simvastatin, simvastatin/ezetimibe and rosuvastatin, respectively, p < 0.001 compared with baseline). No intergroup differences were observed. ConclusionsSimvastatin 40 mg, simvastatin/ezetimibe 10/10 mg and rosuvastatin 10 mg significantly reduced 8-epiPGF2a, oxLDL and Lp-PLA2 activity and mass to a similar extent.

Fast and Fabulous – Patch Clamp Screening of a7 nAChRs

To date, nine alpha (a2 to a10) and three Beta subunits (B2 to B4) have been cloned. Given nAChR putative role in cognition and learning &amp; memory, a number of nAChR allosteric modulators (PAMs) have been synthesized and studied as potential therapeutics for conditions such as Alzheimer's disease, schizophrenia and Parkinson's disease. The gold standard for studying ion channels remains the patch clamp technique. One challenge facing the study of homomeric a7 is its extremely fast desensitization. In this study, we have used HEK cells expressing homomeric nAChR a7 subunits or heteromeric a3B4 nAChR and automated patch clamp devices capable of performing whole cell recordings multiple cells simultaneously with high success rates. Characteristic a7 or a3B4 currents were recorded and could be reliably and repetitively activated by addition of nicotine or acetylcholine. Data will be shown for effect of agonists, PAMs and inihibitors, particularly enhancement of a7 currents by PNU‐120596 and inhibition of a3B4 by mecamylamine, a ganglionic blocker with clinically relevant hypotensive actions.

Abstract 3088: Association between Coronary Plaque Burden as Measured by Multidetector Computed Tomography and Inflammatory Biomarkers in Patients with Acute Chest Pain

Objective: Both the extent of coronary artery disease (CAD) and increased levels of inflammatory markers are associated with cardiovascular risk. However, the association of these markers with different types of atherosclerotic plaque as detected by multidetector computed tomography (MDCT) is unknown. Methods: In this cross-sectional study, we examined consecutive patients who were admitted ith acute chest pain to the ED but had no evidence of ACS during their index hospitalization and 6- month follow up. All patients underwent contrast enhanced 64-slice coronary MDCT. Commercially available assays were used to determine the levels of hs-CRP, matrix metalloproteinase 2 and 9, oxidized low-density lipoprotein (ox-LDL), tumor necrosis factor alpha, interleukin-6, soluble E-selectin, and lipoprotein-associated phospholipase A2 at the time of initial ED triage. Two independent observers determined the extent of CAD (number of coronary segments containing calcified and/or non-calcified plaque). Log-transformed biomarkers associated with the extent of CAD were determined in univariate and multivariable regression analysis (adjusted for age, gender, body mass index, diabetes mellitus, smoking, hypertension, and hyperlipidemia/ use of cholesterol lowering medication). Results: We analyzed inflammatory biomarkers and extent of CAD in 78 patients (mean age: 51.2 years, 42% female). In univariate analysis, hs-CRP (r=0.24, &gt;p=0.02), and ox-LDL (r=0.33, p=0.008) were significantly correlated to the extent of any plaque, and non-calcified plaque, respectively. In multivariable analysis only ox- LDL remained an independent predictor of the extent of non-calcified plaque (beta coefficient: 1.81, p=0.005). The models including traditional risk factors and hs-CRP or ox-LDL explained 34.3%, and 24.5% of the variability of overall, and non-calcified plaque burden, respectively. Conclusions : In this exploratory analysis, we found that in patients with acute chest pain levels of ox-LDL were independent predictors of the extent of non-calcified plaque as detected by MDCT. Further studies are needed to confirm a complementary role of local (plaque) and systemic biomarkers of atherosclerosis to identify subjects at high probability of ACS.

Effect of immunostimulating and anti-inflammatory agents on the biosynthesis of prostaglandins E2, F2 alpha, prostacyclin (PGI2) and thromboxane a2 (TXA2) by mouse embryo fibroblasts in tissue culture.

Experiments performed in cultures of mouse embryo fibroblasts have shown that dexamethasone (10(-6) M) significantly inhibits the production of PGE2 and PGI2 but only moderately inhibits that of PGF2 alpha. Conversely, lipopolysacharide treatment (5 micrograms/ml) markedly stimulated the secretion of PGE2 and PGI2 and only slightly that of PGF2 alpha. In these cultures, the secretion of TXA2 remained unaffected either in the presence of dexamethasone or LPS.

Down-regulation of alpha class glutathione S-transferase by interleukin-1beta in human intestinal epithelial cells (Caco-2) in culture.

The influence of pro-inflammatory cytokines on alpha class glutathione S-transferase A1 and A2 (GSTA1/A2) expression was examined in human colonic epithelial cells (Caco-2) in culture. Dose-dependent reductions in GSTA1/A2 mRNA, protein, and activity levels occurred in Caco-2 cells cultured in conditioned medium (CM) from lipopolysaccharide-stimulated murine monocyte-macrophage cells (RAW 264.7). Neutralizing anti-interleukin-1beta (IL-1beta) antibodies attenuated this repression of GSTA1/A2 expression by CM. Moreover, recombinant human IL-1beta reduced GSTalpha expression at the mRNA, protein, and activity levels in a dose-related fashion. Reduction of GSTA1/A2 mRNA levels by IL-1beta was attenuated by pretreatment with IL-1 receptor antagonist. GSTA1/A2 mRNA half-lives were similar in control and IL-1beta-treated cells, indicating that IL-1beta has no effect on mRNA stability. In reporter gene studies, IL-1beta caused a dose-related reduction of luciferase activity in Caco-2 cells transfected with the full-length GSTA1 promoter-luciferase construct. Using truncated constructs, IL-1beta responsiveness was mapped to a region 286 base pairs upstream to the coding region. Deletion of a hepatic nuclear factor 1 (HNF-1) site in this region abrogated the IL-1beta-mediated repression of GSTA1 promoter activity. These results demonstrate that IL-1beta down-regulates GSTA1/A2 expression in cultured human enterocytes by a transcriptional mechanism involving an HNF-1 site.

Modulatory role of protein tyrosine kinase activation in the receptor-induced contractions of the bovine cerebral artery.

Changes in contractile force were measured during isometric contraction of the bovine middle cerebral artery caused by stimulation of various receptors and by application of high K+, caffeine, and protein kinase C (PKC)-activators. The protein tyrosine kinase (PTK)-inhibitors, such as genistein and tyrphostin, were applied before testing the effect on the contractions or during the maximal plateau of the contraction. The contractions induced by serotonin, prostaglandin F2 alpha, endothelin-1, and thromboxane A2 were significantly and dose-dependently depressed by the PTK-inhibitors (IC50 2-15 microM). In contrast, contractions were significantly augmented by 1 microM pervanadate, an inhibitor of phosphoprotein tyrosine phosphatase. Lineweaver-Burk plotting of the dose-response curves with an increase in inhibitor concentration indicated that the receptor affinity for each agonist remained unchanged in spite of marked depression of the responses. Although the effect was not significant, contractions induced by both high K+ and caffeine were also depressed slightly by PTK-inhibitors in the same range of concentrations used for receptor-induced contractions. Contractions induced by PKC-activators, such as 1-oleoyl-2-acetyl-sn-glycerol and phorbol-12,13-diacetate, were significantly depressed by PTK-inhibitors at concentrations similar to those used for receptor-induced contractions. The results suggest that receptor stimulations which produce sequential activation of phospholipase C and PKC can activate PTK and trigger the so-called "PTK-cascade" causing a sustained or long-lasting contraction similar to the cerebral vasospasm observed clinically.

Discovery of 3-aminobenzyloxycarbonyl as an N-terminal group conferring high affinity to the minimal phosphopeptide sequence recognized by the Grb2-SH2 domain.

The observation that anthranilic acid as N-terminal group produces a dramatic increase of the binding affinity of the phosphopeptide sequence Glu-pTyr-Ile-Asn for the Grb2-SH2 domain was rationalized by molecular modeling. The model, which invokes a stacking interaction between the N-terminal group and the SH2 domain residue Arg alpha A2, was subsequently used to design the 3-aminobenzyloxycarbonyl N-terminal group. The latter confers high affinity (IC50 = 65 nM in an ELISA assay) to the minimal sequence pTyr-Ile-Asn recognized by the Grb2-SH2 domain.

Insoluble glycogen, a metabolizable internal adsorbent, decreases the lethality of endotoxin shock in rats

Insoluble glycogen is an enzymatically modified form of naturally occurring soluble glycogen with a great adsorbing capacity. It can be metabolized by phagocytes to glucose. In this study we used insoluble glycogen intravenously in the experimental endotoxin shock of rats. Wistar male rats were sensitized to endotoxin by Pb acetate. The survival of rats were compared in groups of animals endotoxin shock treated and non-treated with insoluble glycogen. Furthermore, we have determined in vitro the binding capacity of insoluble glycogen for endotoxin, tumour necrosis factor alpha, interleukin-1 and secretable phospholipase A2. Use of 10 mg/kg dose of insoluble glycogen could completely prevent the lethality of shock induced by LD50 quantity of endotoxin in rats. All animals treated survived. Insoluble glycogen is a form of ‘metabolizable internal adsorbents’. It can potentially be used for treatment of septic shock.

THE MOLECULAR CHAPERONE FUNCTION OF α‐CRYSTALLIN IS IMPAIRED BY UV PHOTOLYSIS

Buffer solutions of the lens protein gamma-crystallin and the enzymes aldolase and liver alcohol dehydrogenase became turbid and formed solid precipitate upon exposure to an elevated temperature of 63 degrees C or to UV radiation at 308 nm. When alpha-crystallin was added to the protein solutions in stoichiometric amounts, heat or UV irradiation did not cause turbidity, or turbidity developed much less rapidly than in the absence of alpha-crystallin. Hence, normal alpha-crystallin functioned as a "molecular chaperone," providing protection against both UV and heat-induced protein aggregation. When alpha-crystallin was preirradiated with UV at 308 nm, its ability to function as a chaperone vis-a-vis both UV and heat-induced aggregation was significantly impaired, but only at relatively high UV doses. A major effect of preirradiation of alpha-crystallin was to cause interpeptide crosslinking among the alpha A2 and alpha B2 subunits of the alpha-crystallin macromolecule. In our experiments alpha-crystallin was exposed to UV doses, which resulted in 0.50 and 90% crosslinking as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha-Crystallin samples that were 50% and 90% crosslinked gave chaperone protection, which was increasingly impaired relative to unirradiated alpha-crystallin. The results are consistent with the notion that UV irradiation of alpha-crystallin results in loss of chaperone binding sites.

Activin A and retinoic acid synergize in cyclooxygenase-1 and thromboxane synthase induction during differentiation of J774.1 macrophages.

The murine macrophage cell line J774.1 was used to study the development of prostanoid biosynthesis under the influence of activin A and retinoic acid. Treatment of cells with 3 nM activin A for 48 h increased the biosynthesis of the prostaglandins E2, D2, F2 alpha and thromboxane A2 more than fourfold due to an induction of cyclooxygenase-1 while cyclooxygenase-2 was unaffected. Transforming growth factor-beta acted in a similar way. Retinoic acid, when present alone, was without effect on the total cyclooxygenase products and only slightly changed the pattern of prostanoids. However, when coincubated with activin A, retinoic acid specifically induced the synthesis of thromboxane-A-synthase-specific mRNA and induced an increase in enzyme activity with a synergistic effect on cyclooxygenase-1 protein and mRNA. JunB, but not c-jun, mRNA expression was found under these conditions in addition to a transient c-fos mRNA increase. The combination of activin A and retinoid acid may be regarded as a differentiation model to study the development of cell-specific prostanoid patterns in macrophages and possibly other differentiating cells.

Chaperone activity of alpha-crystallins modulates intermediate filament assembly.

Intermediate filaments are generally regarded as one of the most insoluble and resilient cytoskeletal structures of eukaryotic cells. In extracts from the ocular lens, we noticed an unusually high level of vimentin in a soluble, non-filamentous form. Immunoprecipitation of this soluble vimentin resulted in the co-precipitation of alpha-crystallins. The alpha-crystallins are homologous to the small heat shock proteins (sHSPs) and have recently been identified as molecular chaperones, capable of preventing the heat-induced aggregation of proteins. We find that the alpha-crystallins dramatically inhibit the in vitro assembly of GFAP and vimentin in an ATP-independent manner. This inhibition is also independent of the phosphorylation state of the alpha-crystallin polypeptides and each one of the four polypeptides, either alpha A1-, alpha A2-, alpha B1- or alpha B2-crystallin, are equally effective in this inhibition. Furthermore, we show that alpha-crystallins can increase the soluble pool of GFAP when added to preformed filaments. Electron microscopy demonstrated that alpha-crystallin particles could bind to intermediate filaments in a regular fashion, the spacing coinciding with the molecular length of GFAP. This is the first report, as far as we are aware, of a chaperone being involved in intermediate filament assembly and implicates chaperones in the remodeling of intermediate filaments during development and cell differentiation.

Renal Synthesis of Prostaglandins and Thromboxane Has No Causative Role for Protein-Induced Glomerular Hyperfiltration in Healthy Humans

The study was devised to establish the role of prostaglandin E2, prostaglandin1 alpha and thromboxane A2 in the genesis of glomerular hyperfiltration response to a meat meal in healthy humans. To this end, a group of 8 healthy women was studied 4 times: in the control state (study 1), after a meat meal (study 2), following a meat meal associated with intravenous aspirin (study 3) and finally after a meat meal associated with intravenous aspirin following 2-day pretreatment with oral aspirin (study 4). Urinary excretion of prostaglandins and thromboxane increased during the glomerular hyperfiltration response to a meat meal and was suppressed by aspirin administration which did not suppress the renal hemodynamic response to the meat meal. The data do not support a causal role for prostaglandins and thromboxane A2 in the hemodynamic response to acute protein loading.

Parturitional factors associated with membrane stripping

Our purpose was to determine what factors occurring after digital separation of the chorionic membranes from the lower uterine segment (membrane stripping) are involved in observed clinical changes compared with patients not so treated. Thirty patients were randomly divided among a study population and two control groups to assess uterine contractions and microbiologic, histologic, and biochemical markers associated with parturitional events over a 7-hour time frame. Clinically, an increased frequency of uterine contractile activity was observed among patients in the membrane-stripped group (p < 0.03). There was a significant increase in plasma 13,14-dihydro-15-keto-prostaglandin F2 alpha (p < 0.001) and endocervical phospholipase A2 activity (p < 0.04) among those who underwent membrane stripping. Blood leukocyte counts, sedimentation rates, prostaglandin E2 metabolite concentrations, and fibronectin levels revealed no significant change during the 7-hour study session. Membrane stripping was associated with increases in phospholipase A2 activity and prostaglandin F2 alpha concentrations, indicating a possible correlation with initiation of the cascade of parturitional events.

Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine.

Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.

Regulation of cytosolic pH of cultured mesangial cells by prostaglandin F2 alpha and thromboxane A2

Prostaglandins (PG) and thromboxane A2 (TxA2) have marked vasoactive effects on the renal glomerular microcirculation. Exposure of cultured mesangial cells to PGF2 alpha and TxA2 mimetics results in a rapid elevation of free cytosolic Ca2+ ([Ca2+]i) followed by contraction and cell proliferation. We studied whether other ionic changes mediate these effects of eicosanoids on cells of rat and human origin. Cytoplasmic pH (pHi) was monitored in cells loaded with the fluorescent, intracellularly trapped pH-sensitive probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. PGF2 alpha in rat cells and the TxA2 mimetic U-46619 in human cells induced rapid, dose-dependent cytosolic acidification followed by recovery and net alkalinization mediated by enhanced Na(+)-H+ exchange. The early acidification was also stimulated by ionomycin and Ca2(+)-mobilizing peptides, implicating a Ca2(+)-dependent mechanism. Alkalinization was abolished by removal of extracellular Na+ and by amiloride. Both components of the responses were inhibited by phorbol myristate acetate, which could mimic alkalinization, suggesting a regulatory role of protein kinase C in activation of the Na(+)-H+ exchanger by eicosanoids. Vasoconstrictor arachidonate metabolites may control glomerular cell function by a signaling mechanism centered on concurrent changes of pHi and [Ca2+]i.

Immune-mediated mesangial cell injury—Biosynthesis and function of prostanoids

We studied the formation of cyclo-oxygenase products in a rat model of mesangial cell injury, in order to determine a possible role of prostaglandin E2 (PGE2), prostaglandin I2 (determined as 6-keto-PGF1 alpha and thromboxane A2 (TxA2) in immune-mediated glomerular disease. Selective immune-mediated mesangial cell injury was induced by i.v. administration of a rabbit anti-rat thymocyte antiserum (ATS). Intravenous ATS leads to immune deposits in the mesangium followed by mesangiolysis and the infiltration of polymorphonuclear granulocytes and monocytes. Glomerular TxB2 formation two hours (292 +/- 27 pg/mg/min) and 48 hours (396 +/- 69 pg/mg/min) following antibody was significantly (P less than 0.05) higher compared to animals receiving non-antibody rabbit IgG (TxB2: 2 hr 143 +/- 13; 48 hr 171 +/- 32 pg/mg/min). Treatment with cobra venom factor (CVF) and the reduction of glomerular monocyte infiltration inhibited the increase of glomerular TxB2 formation significantly. Depletion of granulocytes with a rabbit anti-rat granulocyte serum had no effect on glomerular prostanoid formation following ATS. Glomerular PGE2 and 6-keto PGF1 alpha production was not altered following ATS. Inulin clearance in rats with immune-mediated mesangial cell injury was significantly (P less than 0.001) lower at two hours (456 +/- 24 microliters/min/100 g body wt) and 48 hours (433 +/- 54 microliters/min/100 g body wt) compared to their corresponding control animals which were treated with non-antibody IgG (2 hr: 914 +/- 51; 48 hr: 694 +/- 79 microliters/min/100 g body wt).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of phosphorylation on the structure of α-crystallin

Homopolymers were constructed from the highly purified phosphorylated (A1 and B1) and non-phosphorylated (A2 and B2) polypeptides of alpha-crystallin. These were examined using electron microscopy, light scattering, fluorescence spectroscopy and sulphydryl probing to determine the effects of phosphorylation on the structure of alpha-crystallin. Each reconstituted aggregate consisted of uniform particles with circular cross-sections and diameters of 9.3-9.5 nm. Some of these were associated in chain-like structures. The molecular mass of the homopolymers varied from 360-507 kDa; for a single particle, it was estimated to be 216 +/- 10 kDa. Tryptophan residues in the alpha B2 homopolymers were more accessible to the solvent and to quenchers than those in alpha A2. Phosphorylation increased this accessibility in the alpha A homopolymer but decreased it in alpha B. This decrease could be attributed to phosphorylation of the serine adjacent to tryptophan 60 in the B chain. The kinetics of the reaction with DTNB indicated that the single cysteine in the A chain was buried in the alpha A2 homopolymer (k1' = 0.013 min-1) but more accessible in alpha A1 (k1' = 0.046 min-1). It was concluded that phosphorylation significantly alters the conformation of the alpha A subunits but probably has little effect on the B subunits. The alterations do not affect the ability of the subunits to associate into alpha-crystallin-like particles.

Sperm function and structure and seminal plasma prostanoid concentrations in men with IDDM.

Semen from 18 men with insulin-dependent diabetes mellitus (IDDM) aged 20-40 yr was compared with that from 15 age-matched control subjects. Although semen volume, sperm count, and spermatozoal motility were similar in the two groups, semen from diabetic men had significantly greater numbers of abnormal spermatozoa and significantly lower ability to penetrate hamster eggs. Concentrations of prostaglandins E2, F2 alpha, and I2 and thromboxane A2 were significantly elevated in the seminal plasma from semen of diabetic subjects compared with control subjects. These observations indicate the need for a careful assessment of fertility in diabetic men, the mechanisms underlying the abnormalities in spermatozoa, and the relationship of these abnormalities to the increase in prostanoid concentrations in diabetic men.