Post-exposure prophylaxis following consented sexual exposure: impact of national recommendations on user profile, drug regimens and estimates of averted HIV infections.

OBJECTIVES: The aim of the study was to describe the characteristics, impact and outreach of post-exposure prophylaxis (PEP) for sexual exposure in Brazil. METHODS: We used secondary data from the Brazilian Ministry of Health to describe the impact of national guidelines on the frequency of prescription, user profile and antiretroviral regimens. We also estimated the number of potentially averted HIV infections attributable to PEP for consented sexual exposure between 2009 and 2017. RESULTS: A total of 260 457 PEP regimens were prescribed to individuals ≥ 14 years old; 104 613 (40.2%) were prescribed for consented sexual exposure, with an increasing frequency since 2011. Drugs used in PEP regimens underwent significant modifications during the period, reflecting national recommendations. We estimated that there were up to 3138 potentially averted HIV infections attributable to PEP for consented sexual exposure between 2009 and 2017. CONCLUSIONS: In the context of a combined HIV prevention strategy, PEP is still an essential tool for individuals for whom other methods are contraindicated or fail to be applied.

The role of neurotransmitters and neuromodulators in the pathogenesis of cluster headache: a review.

The pathogenesis underlying cluster headache remains an unresolved issue. Although both the autonomic system and the hypothalamus play a central role, the modality of their involvement remains largely unknown. It is, also, unknown why the duration of the pain attacks is so brief and why their onset and termination are abrupt and extremely painful. This review summarizes the evidence to date accumulated in favor of a possible role of anomalies in the metabolism of tyrosine, tryptophan, and arginine in these unresolved issues.

Pathogenesis of chronic cluster headache and bouts: role of tryptamine, arginine metabolism and α1-agonists.

The aim of this study was to explore the possible role of tryptamine in the pathogenesis of chronic cluster headache along with that of adrenaline and noradrenaline (α-agonists) together with arginine metabolism in the origin of cluster bouts. Plasma levels of tyramine, tryptamine, serotonin, 5-hydroxyindolacetic acid, noradrenalin, adrenalin and the markers of arginine metabolism such as arginine, homoarginine, citrulline, ADMA and NMMA, were measured in 23 chronic cluster headache patients (10 chronic cluster ab initio and 13 transformed from episodic cluster) and 28 control subjects. The plasma levels of tyramine, tryptamine, noradrenalin and adrenalin were found several times higher in chronic cluster headache patients compared to controls, whereas the plasma levels of arginine, homoarginine and citrulline were significantly lower. No differences were found in the plasma levels of serotonin, 5-hydroxyindolacetic, ADMA and NMMA between chronic cluster headache patients and control subjects. These results provide support for a role of tryptamine in the pathogenesis of chronic cluster headache and, in particular, in the duration of the cluster bouts. In addition, the low levels of the nitric oxide substrates together with the high levels of noradrenalin and adrenalin suggest an activation of endothelial TAAR1 receptors followed by the release of nitric oxide in the circulation that may constitute the final step of the physiopathology of cluster crisis.

Peripheral neuromodulation in chronic migraine.

Patients with chronic migraines are often refractory to medical treatment. Therefore, they might need other strategies to modulate their pain, according to their level of disability. Neuromodulation can be achieved with several tools: meditation, biofeedback, physical therapy, drugs and electric neurostimulation (ENS). ENS can be applied to the central nervous system (brain and spinal cord), either invasively (cortical or deep brain) or non-invasively [cranial electrotherapy stimulation, transcranial direct current stimulation and transcranial magnetic stimulation]. Among chronic primary headaches, cluster headaches are most often treated either through deep brain stimulation or occipital nerve stimulation because there is a high level of disability related to this condition. ENS, employed through several modalities such as transcutaneous electrical nerve stimulation, interferential currents and pulsed radiofrequency, has been applied to the peripheral nervous system at several sites. We briefly review the indications for the use of peripheral ENS at the site of the occipital nerves for the treatment of chronic migraine.

Genome-wide analysis reveals the patterns of genetic diversity and population structure of 8 Italian local chicken breeds.

The aim of this study was to conduct a genome-wide comparative analysis of 8 local Italian chicken breeds (Ermellinata di Rovigo, Millefiori di Lonigo [PML], Polverara Bianca, Polverara Nera, Padovana, Pepoi [PPP], Robusta Lionata, and Robusta Maculata), all under a conservation plan, to understand their genetic diversity and population structure. A total of 152 animals were analyzed using the Affymetrix Axiom 600 K Chicken Genotyping Array. The levels of genetic diversity were highest and lowest in PML and PPP, respectively. The results of genomic inbreeding based on runs of homozygosity (ROH; FROH) showed marked differences among breeds and ranged from 0.161 (PML) to 0.478 (PPP). Furthermore, in all breeds, short ROH (<4 Mb in length) were more frequent than long segments. Patterns of genetic differentiation, model-based clustering, and neighbor networks showed that most breeds formed nonoverlapping clusters and were clearly separate populations. The 2 Polverara breeds shared a similar genetic background and showed the lowest genetic differentiation in comparison with purebred lines; the local populations showed separated groups. PPP and PML were closer to the group of the purebred broiler lines (BRSA, BRSB, BRDA, and BRDB). Six genomic regions are presented as hotspots of autozygosity among the Italian chicken breeds, with candidate genes involved in multiple morphological phenotypes as breast muscle, muscle dry matter content, and body weight. This study is the first exhaustive genome-wide analysis of the diversity of these Italian local chickens from Veneto region. We conclude that breeds have conserved authentic genetic patterns. The results are of significant importance because they will help design and implement conservation strategies. In fact, the conservation of these breeds may also have positive impacts on the local economy, niche traditional markets, and offering a source of high-quality products to consumers. In this context, genomic information may play a crucial role in the management of local breeds.

The evolution of South American endemic canids: a history of rapid diversification and morphological parallelism.

The origin of endemic South American canid fauna has been traditionally linked with the rise of the Isthmus of Panama, suggesting that diversification of the dog fauna on this continent occurred very rapidly. Nevertheless, despite its obvious biogeographic appeal, the tempo of Canid evolution in South America has never been studied thoroughly. This issue can be suitably tackled with the inference of a molecular timescale. In this study, using a relaxed molecular clock method, we estimated that the most recent common ancestor of South American canids lived around 4 Ma, whereas all other splits within the clade occurred after the rise of the Panamanian land bridge. We suggest that the early diversification of the ancestors of the two main lineages of South American canids may have occurred in North America, before the Great American Interchange. Moreover, a concatenated morphological and molecular analysis put some extinct canid species well within the South American radiation, and shows that the dental adaptations to hypercarnivory evolved only once in the South American clade.

Mitochondrial diversity of Yoruba and Fulani chickens: A biodiversity reservoir in Nigeria.

Poultry are the most widely distributed type of livestock in Nigeria. Indigenous chickens are extremely common throughout the country. Indeed, approximately 83 million chickens are raised in extensive systems and 60 million in semi-intensive systems. To provide the first comprehensive overview of the maternal lineages in Southwest Nigeria, we analyzed 96 mitochondrial DNA control region sequences from 2 indigenous chicken ecotypes: Fulani and Yoruba. All samples belonged to the most frequent haplogroup (E) in Africa and Europe and showed noticeably low haplotype diversity. Although only 11 different haplotypes were detected, with 2 of them never found before in Nigeria, the presence of unique sequences among our indigenous samples testified to their status as an important genetic resource to be preserved. Furthermore, a total of 7,868 published sequences were included in the comparative analysis, which revealed an east-west geographic pattern of haplogroup distribution and led to the conclusion that the gene flow from Southeastern Asia mainly involved one mitochondrial clade. Moreover, owing to the extensive genetic intermixing among Nigerian chickens, conservation efforts are required to safeguard the extant mitochondrial variability in these indigenous ecotypes and establish future improvement and selection programs.

Molecular methods for cost-efficient monitoring of HAB (harmful algal bloom) dinoflagellate resting cysts.

Cyst abundance and identity are essential for understanding and predicting blooms, and for assessing the dispersal of toxic target dinoflagellate species by natural or human mediated ways, as with ballast waters. The aim of this study was to apply rapid, specific and sensitive qPCR assays to enumerate toxic dinoflagellate cysts in sediment samples collected from Adriatic harbours. The molecular standard curves of various target species allowed obtaining the rDNA copy number per cyst. The analytical sensitivity for specific standard curves was determined to be 2 or 10 rDNA copies per reaction. The abundance varied in the range of 1-747 dinoflagellate cysts g-1 dry weight. The assays showed greater sensitivity as compared to counts by light microscopy. This qPCR method revealed a powerful tool for the quantification of cysts from toxic dinoflagellate resting stages in sediment samples from Adriatic ports.

Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR.

AIMS: The ichthyotoxic species Prymnesium parvum (Haptophyceae) is difficult to quantify in a microscopy-based monitoring programme, because the cells are very small, fragile and their morphology can be distorted by the use of fixatives. In the attempt to overcome these problems, a real-time PCR-based method for the rapid and sensitive identification and quantification of P. parvum was developed. METHODS AND RESULTS: A quantitative real-time PCR assay was optimized with primers designed on the internal transcribed spacer 2 rDNA region of P. parvum. This PCR assay was specific, showing no amplification of DNA extracted from closely related species, and sensitive. Moreover, this method was able to detect and reliably quantify P. parvum cells in preserved environmental samples artificially spiked with known amounts of cultured cells. CONCLUSIONS: Considering the specificity, sensitivity and applicability to preserved environmental samples, this method may be a useful tool for the monitoring of this toxic species. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR method described in this study may represent a progress towards the rapid detection and quantification of P. parvum cells in water-monitoring programmes, allowing the early application of strategies to control bloom events, such as the use of clay minerals.

A distinctive form of human chorionic gonadotropin beta-subunit-like material produced by cervical carcinoma cells.

The DoT and CaSki human cervical carcinoma cell lines ectopically produce material immunologically similar to the beta-subunit of human chorionic gonadotropin (hCG beta). Culture fluids were analyzed by gel filtration chromatography and radioimmunoassay (RIA) using (a) antiserum directed to conformation-specific (core-directed) determinants not involving the carboxyl-terminal peptide (CTP) in hCG beta purified from urinary hCG (i.e., standard hCG beta) or (b) antiserum directed to the CTP in standard hCG beta. CTP-directed RIA recognized a peak of hCG beta-like immunoreactive material that eluted in the same position as standard hCG beta. However, core-directed RIA recognized additional hCG beta-like material (i.e., ectopic beta-II), most of which eluted before standard hCG beta. CaSki cells were incubated with [3H]mannose, [3H]proline, and [3H] leucine, and the spent medium was immunoprecipitated and analyzed by gel electrophoresis. Several labeled peaks were detected in the lane from the anti-hCG beta X Sepharose immunoprecipitate, one of which corresponded in mobility to standard hCG beta, with two more intense components migrating at higher apparent molecular weights. Carboxypeptidase Y digestion released only 0.2 mol equivalents each of [3H]proline and [3H]leucine from the labeled CaSki material immunoprecipitated with anti-hCG beta X Sepharose, compared to 1 mol equivalent each in similar analysis of standard hCG beta. These findings were consistent with the absence of the 4-carboxy-terminal amino acids from 80% of the hCG beta-like immunoreactive material secreted by CaSki cells. The affinity purified ectopic beta-II failed to combine with standard hCG alpha under conditions in which combination of standard hCG beta with standard hCG alpha was essentially complete. Neither aggregation nor proteolytic degradation was the cause of failure of ectopic beta-II to combine with hCG alpha. We conclude that both the DoT and CaSki cervical carcinoma cell lines secrete a distinctive form of hCG beta-like material, ectopic beta-II. Lack of recognition by CTP-directed antisera and amino acid analysis suggest that ectopic beta-II may lack the CTP, despite its apparent larger size relative to standard hCG beta.

N-acetylgalactosamine glycosylation of MUC1 tandem repeat peptides by pancreatic tumor cell extracts.

Synthetic peptides corresponding to the human mucin MUC1 tandem repeat domain (20 residues) were glycosylated in vitro by using UDP-N-[3H]acetyl-D-galactosamine (GalNAc) and lysates of pancreatic tumor cell lines. Results obtained with peptides of different lengths (from one to five repeats) suggest that increasing the number of tandem repeats has neither a positive nor a negative effect on the density of glycosylation along the MUC1 tandem repeat protein backbone. Purified glycopeptides were sequenced on a gas-phase sequencer, and glycosylated positions were determined by measuring the incorporated radioactivity in fractions collected following each round of Edman degradation. The results showed that two of three threonine residues on the MUC1 tandem repeat peptides were glycosylated by pancreatic tumor cell lysates at the following positons: GVTSAPDTRPAPGSTAPPAH (underlined T indicates position of GalNAc attachment). None of the serine residues were glycosylated. Determination of the mass of the glycopeptides by mass spectrometry confirmed that a maximum of two molecules of GalNAc were covalently linked to each 20-residue repeat unit in the peptides. The data presented here show that acceptor substrate specificity of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase detected in lysates of pancreatic and breast tumor cell lines is identical and is limited to some but not all threonines in the MUC1 tandem repeat peptide sequence. The influence of primary amino acid sequence on acceptor substrate activity was evaluated by using several peptides that contain single or double amino acid substitutions (relative to the native human MUC1 sequence). These included substitutions in the residues that were glycosylated and substitutions of the surrounding primary amino acid sequence. The results of these studies suggest that primary amino acid sequence, length, and relative position of the residue to be glycosylated dramatically affect the ability of peptides to serve as acceptor substrates for the UDP-GalNAc:polypeptide N-acetylgalatosaminyltransferase.

Purification and properties of ribonucleases from human urine.

The two major ribonuclease (EC 3.1.27.5) present in normal human urine have been highly purified and extensively characterized for their enzymatic, physical, chemical and structural properties. One of the enzymes, RNAase C, is a glycoprotein which exhibits a pH optimum of 8.5 with RNA as the substrate and preferentially degrades the synthetic homoribopolymer poly(C). This enzyme is resolved into multiple components by column electrofocusing. However, prior treatment with neuraminidase results in a single form of RNAase C with an isoelectric point of 10.4, indicating that the charge heterogeneity is the result of variability in sialic acid content. Amino acid composition and NH2- and COOH-terminal sequence analyses of RNAase C show that this enzyme is very similar to mammalian pancreatic RNAases; the data indicate a peptide chain of 126 amino acid residues and a 33% carbohydrate content. The second enzyme isolated from urine, termed RNAase U, is also a glycoprotein which has a pH optimum of 7.0 with RNA as substrate and is virtually inactive against poly(C). RNAase U lacks sialic acid and focuses as a single component with a highly basic isoelectric point of greater than pH 11.0. The NH2- and COOH-terminal sequences of RNAase U show little homology with the pancreatic RNAases. However, the amino acid composition of this enzyme indicates it is very similar to human spleen RNAase.

[Occupational differences in exposure to hazardous work conditions]. / Differenze per professione nelle condizioni di lavoro nocive.

BACKGROUND: Few studies have been aimed at describing organizational and psychosocial conditions of the Italian workforce by occupational group, and they have been mainly conducted within specific occupations. OBJECTIVES: The present study aimed at identifying specific groups of occupations which have unfavourable profiles from the point of view of exposure to specific organizational factors and psychosocial risks, and to physical, chemical and ergonomic risks, and analyzing their distribution by worker age. METHODS: The analysis was conducted on a sample of 4,195 workers in the Piedmont Region who were members of the CGIL Trade Union (Italian General Confederation of Labour), who answered a self-administered questionnaire in 2000, aimed at assessing chemical, physical, and ergonomic risks, accidents, and psychosocial factors connected with work organization and work tasks. Psychosocial risks were assessed via three scales aimed at measuring the degree of control, psychophysical demands, and worker satisfaction. The proportion of workers exposed to the above mentioned risks was analysed according to occupational group. This group was then compared with all other groups taken together, according to prevalence of high strain condition (combination of high demand and low control) and HSUR condition (High Strain Unfairly Rewarded; combination of high strain and low satisfaction). RESULTS: Among males aged 25-44 years, restricted to the occupation groups with more than ten workers in high strain condition, significantly higher proportions of stress were observed in leather workers and shoemakers, paper factory workers, rubber workers, crane and bridge crane operators, plastic workers, painters, transport drivers and carpenters. For many of these groups, excesses were confirmed for the HSUR condition. Among subjects aged over 44 years, a higher risk for high strain was confirmed in rubber workers, transport drivers and carpenters. In addition, machine tool operators, assembly line and mechanical workers in this age group were exposed to higher risk of stress. In younger women there was a significantly higher risk of both high strain and HSUR conditions in workers employed in the rubber, plastic and the food industries, and in machine tool workers. Such excess risk was confirmed in the latter three categories also among older women. In general, the proportion of male workers classified as working in high strain conditions decreased as age increased, while in women it remained stable. CONCLUSIONS: The study allowed evaluation of the differential impact of exposure to physical, chemical, and psychosocial risk factors among occupational groups, thus contributing to the identification and classification of exhausting jobs. The results further showed, particularly among men, a smaller proportion of older workers, compared to younger ones, exposed to harmful physical, chemical, ergonomic and psychosocial conditions, indicating a possible propensity of companies to adapt workplace conditions, organization and internal mobility to worker age.

Event-based prospective memory in patients with Parkinson's disease: the effect of emotional valence.

The present study investigated the effect of Parkinson's disease (PD) on prospective memory (PM) tasks by varying the emotional content of the PM actions. Twenty-one older adults with PD and 25 healthy older adults took part in the present study. Participants performed three virtual days in the Virtual Week task. On each virtual day, participants performed actions with positive, negative or neutral content. Immediately following each virtual day, participants completed a recognition task to assess their retrospective memory for the various PM tasks. PD patients were less accurate than the control group at both PM accuracy and recognition task accuracy. The effect of emotional valence was also evident, indicating that all participants were more accurate on positive PM tasks than both negative and neutral. This study confirmed PM impairment in PD patients and extended previous research showing how positive emotional stimuli can influence PM performance.

O-glycosylation of the alpha-subunit does not limit the assembly of chorionic gonadotropin alpha beta dimer in human malignant and nonmalignant trophoblast cells.

Biosynthetic experiments were carried out in cultures of human malignant trophoblast cells (the JAR cell line) and in explants of normal first trimester human placental tissue to test the hypothesis that the O-glycosylation of the glycoprotein hormone alpha-subunit at Thr-39 regulates the assembly of the CG alpha beta dimer. This modification of alpha has been shown to ablate its ability to combine in vitro with the beta-subunit of bovine LH and might explain why JAR cells and placental explants secrete uncombined alpha- and beta-subunits in addition to the hCG alpha beta dimer. We have previously detected an O-linked carbohydrate chain at Thr-39 in preparations of secreted free alpha-subunit, but not dimer CG alpha from JAR culture medium. We report here evidence that the O-glycosylation of alpha does not regulate the biosynthetic assembly of the hCG dimer in cultures of JAR choriocarcinoma cells or first trimester placental explants. The intracellular precursor forms of alpha and beta that accumulate in the endoplasmic reticulum and combine in that compartment are not yet modified with O-linked carbohydrate, as determined by measurements of their [3H]galactosamine content after biosynthetic labeling of amino sugars with [3H]glucosamine. Furthermore, only half of the free alpha-subunit secreted by JAR cells and less than 10% of free alpha secreted by 10-week-old placental explants received the O-linked chain. This was shown by determining the ratio of the unglycosylated and glycosylated forms of the tryptic peptide from free alpha that contains the O-glycosylation site (residues 36-42). Based on these findings, we make the following conclusions. 1) O-Glycosylation of alpha-subunit is a late event in the secretory pathway of trophoblasts compared to the rapid combination in the rough endoplasmic reticulum of hCG subunit precursors to form alpha beta dimer. 2) Association of alpha with beta precludes the subsequent addition of the glycan to alpha at Thr-39. 3) The alpha molecules that fail to combine with beta in the endoplasmic reticulum are substrates for the later addition of O-linked carbohydrate, presumably in the Golgi complex, but only a fraction of the free alpha molecules are modified with O-linked carbohydrate.

The effect of carbonyl cyanide trifluoromethoxyphenylhydrazone and methylamine on the processing and secretion of the glycoprotein hormone chorionic gonadotropin by human choriocarcinoma cells.

Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), a protonophore, and methylamine, a weak base, agents that dissipate hydrogen gradients across cellular membranes, were used to probe the coupling of hydrogen gradients to the processing and secretion of the glycoprotein hormone hCG by human choriocarcinoma cells (JAR) in culture. Both drugs disrupted the processing of asparagine-linked oligosaccharides such that the secreted hCG forms contained mostly high mannose rather than complex oligosaccharide chains. As the concentrations of FCCP were increased above 1 microgram/ml and those of methylamine above 12.5 mg/ml, the secretion of the labeled hCG dimer and free alpha-subunit was progressively inhibited. Both FCCP and methylamine also inhibited the incorporation of [35S] methionine and [3H]mannose into hCG subunits. Nevertheless, the inhibition of secretion was clearly apparent as an intracellular accumulation of the hCG subunit precursors in spite of the diminished incorporation of radioactive substrates. The intracellular hCG precursors that accumulated in the drug-treated cells contained predominantly Man8-9GlcNAc2 units, structures characteristic of glycoproteins localized in the endoplasmic reticulum. Both FCCP and methylamine inhibited hCG secretion at concentrations that did not lower the cellular content of ATP. We postulate on the basis of these results that a hydrogen gradient across the membrane either of the rough endoplasmic reticulum or the transitional vesicle is coupled to the rough endoplasmic reticulum to Golgi translocation step such that dissipation of the proton gradient blocks the secretion of hCG.

Conformational intermediates in the production of the combinable form of the beta-subunit of chorionic gonadotropin.

Human trophoblastic cells synthesize and secrete hCG as well as uncombined forms of the alpha- and beta-subunits of hCG. We have previously reported that the rate-limiting step in alpha beta-dimer assembly in cultured JAR choriocarcinoma cells is a conformational change in beta-subunit accompanied by the formation of intramolecular disulfide bonds. We now report on the intermediate steps in the acquisition of this combinable conformation by the beta-subunit. The earliest biosynthetically labeled form of beta detected in JAR cells is a precursor termed p beta 1 that lacks at least one of the intramolecular disulfide bonds found in mature beta-subunit, that does not combine with alpha-subunit, and that does not react with a monoclonal antibody specific for free beta. The p beta 1 precursor rapidly assumes (within 5 min) a new conformation termed p beta 2 that, in contrast to p beta 1, migrates more slowly on nonreduced sodium dodecyl sulfate-polyacrylamide gels, combines with alpha to form the hCG dimer, and reacts with the monoclonal anti-free beta antibody. Pulse-chase kinetic experiments support the following sequence of events: p beta 1----uncombined p beta 2----combined p beta 2. The transition of p beta 1 to uncombined p beta 2 involves the formation of at least one intramolecular disulfide bond coincident with the conformational shift of the p beta molecule. Furthermore, treatment of the nonreduced subunits with trypsin releases a [35S]cysteine-labeled peptide from p beta 1, but not from either form of p beta 2. This peptide presumably contains one of the two crucial cysteine residues that participate in forming the disulfide bond that distinguishes p beta 1 from the p beta 2 forms. Dimer p beta 2 differs from both p beta 1 and uncombined p beta 2 in that it contains an O-linked N-acetylgalactosamine, which represents the first step in the formation of the O-linked glycans of beta-subunit. Dimer p beta 2 is, therefore, the most fully processed and kinetically the latest of the three p beta forms that appear in JAR cell lysates. We conclude that formation of an appropriate array of intramolecular S-S bonds accompanies the acquisition of a combinable conformation of beta-subunit, and we have identified intermediate steps in the pathway leading to this conformational change. The data suggest that it is the achievement of this conformation by beta-subunit that limits the alpha beta combination reaction rather than the amount or conformation of alpha-subunit.

Identification and characterization of subpopulations of the free alpha-subunit that vary in their ability to combine with chorionic gonadotropin-beta.

The free (uncombined) alpha-subunit of hCG is secreted in excess over alpha beta dimer from both malignant and nonmalignant trophoblast cells and is secreted ectopically from a variety of other malignant cell types. The free alpha-subunits from various sources are distinguishable from those that combine because they migrate more heterogeneously and more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than dimer alpha. We have previously identified three posttranslational modifications that may contribute to the altered mobility of the free alpha-subunit and to its inability to combine with the beta-subunit: 1) preferential phosphorylation of the free alpha-subunit, 2) O-glycosylation of free alpha, and 3) differences in the processing of the asparagine-linked oligosaccharides between the free and combinable forms. We have purified three populations of the alpha-subunit from the JAR choriocarcinoma cell line and from ChaGo, a bronchogenic carcinoma cell line that ectopically synthesizes only the alpha-subunit, in order to identify the posttranslational modifications that contribute to the altered mobility on SDS-PAGE. Fractionation of the oligosaccharides released from the alpha forms with peptide N-glycosidase has shown that the faster migrating alpha forms on SDS-PAGE have less completely processed oligosaccharide chains. Twenty-two to 25% of the JAR free alpha and 35-41% of the ChaGo alpha forms that migrate the fastest on SDS-PAGE recombine with beta in an in vitro recombination assay under conditions where 62% of the dimer alpha form recombines. In contrast, only 5-12% and 16-21% of the JAR free alpha and ChaGo alpha forms, respectively, that migrate the slowest on SDS-PAGE recombine with beta. The form of JAR free alpha least capable of combining with beta contains on O-linked glycan on Thr-39. This same site is a substrate for phosphorylation by JAR cells. However, most of ChaGo alpha fails to recombine with beta even though ChaGo alpha contains little O-linked carbohydrate. These results suggest that the larger asparagine-linked complex glycans on the slower migrating alpha forms are the major limiting factor for subunit combination. Although these modifications may not be rate limiting for combination in the rough endoplasmic reticulum, they may prevent dimerization of the free subunits later in the secretory pathway.

Design of substrate-site-directed inhibitors of adenylate kinase and hexokinase. Effect of substrate substituents on affinity on affinity for the adenine nucleotide sites.

Nineteen derivatives of adenosine 5'-phosphate (AMP) bearing acylaminomethyl, acetoxy, or alkylaminomethyl substituents on the phosphate-ribose bridge (5' and O-5' positions) of AMP together with 2',3'-O-ethylidene, 2',3',-O-isopropylidene, and 2',3'-di-O-acetyl derivatives of AMP have been synthesized. Their substrate and/or competitive inhibitor properties with pig rabbit muscle AMP kinases indicate that all the substituents except 2',3'-O-ethlidene with the pig enzyme permitted binding of AMP at its enzymic site. Determination of enzyme-inhibitor dissociation constants showed that several compounds with substituents on the ribose-phosphate bridge bind as well or better than AMP. The affinity is ascribed in part to interaction between substituents and a lipophilic region of the enzymes adjacent to the ribose-phosphate bridge in the enzyme-AMP complexes. The enzyme-inhibitor dissociation constants reveal a structural dissimilarity between the pig and rabbit enzymes in the vicinity of the lipophilic region. The substrate and inhibitor properties of eight ATP derivatives gave evidence that affinity of ATP for its substrate site on the AMP kinases is compatible with acetyl- or chloroacetylaminomethyl groups at the phosphate-ribose bridge or with 2',3'-O-ethylidene or isopropylidene residues. The yeast hexokinase-ATP complex tolerated an acetylaminomethyl group at C-5' or a benzoylaminomethyl group adjacent to O-5'. The present findings regarding substituent tolerance could be used in the design of adenine nucleotide site-directed irreversible inhibitors.