Extended C-band tunable multi-channel InP-based coherent receiver PICs.

Fully integrated monolithic, multi-channel InP-based coherent receiver PICs and transceiver modules with extended C-band tunability are described. These PICs operate at 33 and 44 Gbaud per channel under dual polarization (DP) 16-QAM modulation. Fourteen-channel monolithic InP receiver PICs show integration and data rate scaling capability to operate at 44 Gbaud under DP 16-QAM modulation for combined 4.9 Tb/s total capacity. Six channel simultaneous operation of a commercial transceiver module at 33 Gbaud is demonstrated for a variety of modulation formats including DP 16-QAM for >1.2Tbit/s aggregate data capacity.

1.12 Tb/s superchannel coherent PM-QPSK InP transmitter photonic integrated circuit (PIC).

In this work, a 10-wavelength, polarization-multiplexed, monolithically integrated InP coherent QPSK transmitter PIC is demonstrated to operate at 112 Gb/sec per wavelength and total chip superchannel bandwidth of 1.12 Tb/s. This demonstration suggests that increasing data capacity to multi-Tb/s per chip is possible and likely in the future.

Lymphocyte transformation induced by autologous cells. IV. Human T-lymphocyte proliferation induced by autologous or allogeneic non-T lymphocytes.

An autologous mixed lymphocyte reaction was demonstrated between T and non-T lymphocytes. Sheep erythrocyte rosetting was used to separate human lymphocytes into T and non-T lymphoid preparations. Non-T lymphocytes stimulated the proliferation of autologous T lymphocytes. The cell in this preparation that was most stimulatory had the characteristics of a K lymphocyte. The allogeneic mixed lymphocyte reaction was also shown to reflect the proliferation of T lymphocytes stimulated by allogeneic non-T lymphocytes. Proliferation of T lymphocytes in the allogeneic mixed lymphocyte culture probably reflects a response to both foreign histocompatibility determinants and determinants present on non-T lymphocytes. It is suggested that the proliferative response of T lymphocytes to autologous non-T lymphocytes may be a step in the process by which T lymphocytes regulate immunity.

Immunological studies of Aging. III. Cytokinetic basis for the impaired response of lymphocytes from aged humans to plant lectins.

The basis for the age-associated defect in the response of lymphocytes to plant lectins has been studied. Using three independent assays we have shown that the number of mitogen-responsive cells is markedly reduced in lymphocyte preparations from old persons. In addition, studies using colchicine bloock and thymidine pulse techniques have revealed a failure of mitogen-responsive cells from old persons to expand into a proliferating pool of lymphocytes as is observed when lymphocytes from young persons are cultured with phytohemagglutinin. Thus, the impaired response of lymphocytes from old persons to mitogens is attributable to a reduced number of mitogen responsive cells and their failure to undergo clonal expansion.

The cellular basis of the impaired autologous mixed lymphocyte reaction in patients with systemic lupus erythematosus.

The autologous mixed lymphocyte reaction is impaired in patients with systemic lupus erythematosus. This is because nonthymus-derived (T)-lymphocyte preparations from such patients do not stimulate autologous T-lymphocyte proliferation normally. This defect may explain the impaired generation of suppressor activity in systemic lupus erythematosus and thereby the occurrence of autoantibodies in this disease.

Induction of suppressor activity in the autologous mixed lymphocyte reaction and in cultures with concanavalin A.

T lymphocytes that are activated in the autologous mixed lymphocyte reaction (MLR) have suppressor activity. Concanavalin A (Con A) augments the suppressor activity generated in cultures containing both T and non-T lymphocytes and can induce suppressor activity in T-lymphocyte preparations that contain too few (10%) non-T cells to generate a significant autologous MLR. However, when such T-lymphocyte preparations are further depleted of adherent cells and contain less than 2% non-T cells, Con A fails to induce suppressor activity. These findings support the concept that an autologous MLR may play an important role in generation of suppressor cells by Con A.

Direct correlation between a highly damped modulation response and ultra low relative intensity noise in an InAs/GaAs quantum dot laser.

We describe modulation responses and relative intensity noise (RIN) spectra of an InAs/GaAs quantum dot laser operating near 1300 nm. A very large nonlinear gain compression coefficient yields a highly damped modulation response with a maximum 3 dB bandwidth of ~6.5 GHz and flat RIN spectra which reach as low a level as -158/-160 dB/Hz at frequencies up to 10 GHz.

A plastid terminal oxidase comes to light: implications for carotenoid biosynthesis and chlororespiration.

Inactivation of a plastid located quinone-oxygen oxidoreductase gene in the immutans Arabidopsis mutant leads to a photobleached phenotype because of a lack of photoprotective carotenoids. Inactivation of the corresponding gene in the ghost tomato mutant leads to a similar phenotype in leaves and to carotenoid deficiency in petals and ripe fruits. This plastid terminal oxidase (the first to be cloned and biochemically characterized) resembles the mitochondrial cyanide-insensitive alternative oxidase. Here, we propose a model integrating this novel oxidase as a component of an electron transport chain associated to carotenoid desaturation, as well as to a respiratory activity within plastids.

Biochemistry and molecular biology of chromoplast development.

Plant cells contain a unique class of organelles, designated the plastids, which distinguish them from animal cells. According to the largely accepted endosymbiotic theory of evolution, plastids are descendants of prokaryotes. This process requires several adaptative changes which involve the maintenance and the expression of part of the plastid genome, as well as the integration of the plastid activity to the cellular metabolism. This is illustrated by the diversity of plastids encountered in plant cells. For instance, in tissues undergoing color changes, i.e., flowers and fruits, the chromoplasts produce and accumulate excess carotenoids. In this paper we attempt to review the basic aspects of chromoplast development.

A class-I intron in a cyanelle tRNA gene from Cyanophora paradoxa: phylogenetic relationship between cyanelles and plant chloroplasts.

Cyanelles are photosynthetic organelles which are considered as intermediates between cyanobacteria and chloroplasts, and which have been found in unicellular eukaryotes such as Cyanophora paradoxa. The nucleotide sequence of a 667-bp region of the cyanelle genome from Cyanophora paradoxa containing genes coding for tRNA(UUCGlu) and tRNA(UAALeu) has been determined. The gene coding for tRNA(UAALeu) is split by a 232-bp intron which has a secondary structure typical for class-I structured introns and which is closely related to the intron located in the corresponding gene from liverwort and higher plant chloroplasts. It appears therefore that these tRNA(UAALeu) genes are all derived from one common ancestral gene which already contained a class-I intron.

Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating zeta-carotene desaturation.

We have cloned a cDNA from the plant Capsicum annuum which encodes a novel enzyme mediating the dehydrogenation of zeta-carotene and neurosporene to lycopene when expressed in E. coli cells accumulating zeta-carotene or neurosporene. This enzyme is unable to dehydrogenate either phytoene or lycopene. The deduced amino acid sequence suggests that this cDNA encodes a polypeptide whose mature size is ca. 59 kDa and which is synthesized as a precursor with a NH2-terminal extension resembling transit peptides for plastid targeting. Sequence comparison reveals 33-35% similarity with previously cloned plant or cyanobacterial phytoene desaturases. In contrast, only limited sequence similarity is found with a zeta-carotene desaturase from the cyanobacterium Anabaena.

High level expression of full-length estrogen receptor in Escherichia coli is facilitated by the uncoupler of oxidative phosphorylation, CCCP.

The expression of high levels of full-length human estrogen receptor alpha (hERalpha) in Escherichia coli has proven difficult. We found that expression of the ER DNA binding domain is highly toxic to E. coli, resulting in rapid loss of the expression plasmid. Using a tightly regulated arabinose expression system and the antibiotic Timentin, we were able to overcome ER toxicity and express substantial levels of ER. The expressed ER exhibited protease cleavage at a single site near the N-terminus of the hinge region. Of the many measures we tested to eliminate ER cleavage, only addition of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely blocked intracellular proteolysis of the ER. Using CCCP and our expression methods, full-length FLAG epitope-tagged hERalpha (fER) was expressed in E. coli at approximately 1 mg/l. The fER was purified to homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified full-length bacterial fER binds 17beta-estradiol with the same affinity as hER expressed in human cells (K(D) approximately 0.5 nM). At high concentrations of fER (20 nM), a bell-shaped estrogen binding curve with a Hill coefficient of 1.7 was seen. Bacterially-expressed fER exhibits a reduced affinity for the estrogen response element (ERE). Anti-FLAG antibody restores high affinity binding of the fER to the ERE, suggesting that impaired dimerization may be responsible for the reduced affinity of bacterially-expressed fER for the ERE. The use of Timentin and CCCP may provide a general method for high level bacterial expression of steroid/nuclear receptors and other proteins important in hormone action.

cDNA cloning of the E1 alpha subunit of the branched-chain alpha-keto acid dehydrogenase and elucidation of a molecular basis for maple syrup urine disease.

We have cloned cDNAs encoding human and rat liver BCKDH E1 alpha subunits and deduced the primary structure of the mature protein. The sequences of the cDNA and protein are highly conserved between the two species. Significant sequence similarity has also been found between human BCKDH and PDH E1 alpha subunits. We have studied the molecular basis of MSUD by determining the enzyme activity and levels of BCKDH protein and mRNA, and by enzymatic amplification and sequencing of BCKDH E1 alpha-specific mRNA, from an MSUD patient and his parents. Different mutant alleles were identified in the two parents. The patient was a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father and an allele containing a defect in regulation from the mother. Our results demonstrate that a case of MSUD was caused by structural and regulatory mutations involving the E1 alpha subunit.

Extracorporeal CO2-removal with a heparin coated artificial lung.

Treatment of severe acute respiratory failure with extracorporeal gas exchange necessitating near complete systemic anticoagulation requires a delicate balance to be maintained between disseminated intravascular coagulation and hemorrhagic complications. The present study describes our first experience using a heparin coated extracorporeal artificial lung and circuitry during clinical extracorporeal CO2 removal. In spite of a partial thromboplastin time and activated clotting time within or close to the normal range, neither laboratory evidence for disseminated intravascular coagulation induced by the extracorporeal circuit nor thrombi in the pulmonary vasculature were found. Scanning electron microscopy of the heparin coated hollow fiber gas exchanger demonstrated only minor deposits on the surface. Use of a heparin coated artificial lung may enhance the margin of safety of extracorporeal gas exchange and ultimately broaden its indications.

Regulation of the branched-chain alpha-ketoacid dehydrogenase and elucidation of a molecular basis for maple syrup urine disease.

The hepatic branched-chain alpha-ketoacid dehydrogenase complex plays an important role in regulating branched-chain amino acid levels. These compounds are essential for protein synthesis but toxic if present in excess. When dietary protein is deficient, the hepatic enzyme is converted to the inactive, phosphorylated state to conserve branched-chain amino acids for protein synthesis. When dietary protein is excessive, the enzyme is in the active, dephosphorylated state to commit the excess branched-chain amino acids to degradation. Inhibition of protein synthesis by cycloheximide, even when the animal is starving for dietary protein, results in activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex to prevent accumulation of branched-chain amino acids. Likewise, the increase in branched-chain amino acids caused by body wasting during starvation and uncontrolled diabetes is blunted by activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex. The activity state of the complex is regulated in the short term by the concentration of branched-chain alpha-ketoacids (inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase) and in the long term by alteration in total branched-chain alpha-ketoacid dehydrogenase kinase activity. cDNAs have been cloned and the primary structure of the mature proteins deduced for the E1 alpha subunit of the human and rat liver branched-chain alpha-ketoacid dehydrogenase complex. The cDNA and protein sequences are highly conserved for the two species. Considerable sequence similarity is also apparent between the E1 alpha subunits of the human branched-chain alpha-ketoacid dehydrogenase complex and the pyruvate dehydrogenase complex. Maple syrup urine disease is caused by an inherited deficiency in the branched-chain alpha-ketoacid dehydrogenase complex. The molecular basis of one maple syrup urine disease family has been determined for the first time. The patient was found to be a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father, and an allele which is not expressed from the mother. The only known animal model for the disease (Polled Hereford cattle) has also been characterized. The mutation in these animals introduces a stop codon in the leader peptide of the E1 alpha subunit, resulting in premature termination of translation. Two thiamine responsive patients have been studied. The deduced amino acid sequences of the mature E1 alpha subunit and its leader sequence were normal, suggesting that the defect in these patients must exist in some other subunit of the complex. 3-Hydroxyisobutyrate dehydrogenase and methylmalonate-semialdehyde dehydrogenase, two enzymes of the valine catabolic pathway, were purified from liver tissue and characterized.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of branched-chain alpha-ketoacid dehydrogenase complex by covalent modification.

The branched-chain alpha-ketoacid dehydrogenase complex, like the pyruvate dehydrogenase complex, is an intramitochondrial enzyme subject to regulation by covalent modification. Phosphorylation causes inactivation and dephosphorylation causes activation of both complexes. The branched-chain alpha-ketoacid dehydrogenase kinase, believed distinct from pyruvate dehydrogenase kinase, is an integral component of the branched-chain alpha-ketoacid dehydrogenase complex and is sensitive to inhibition by branched-chain alpha-ketoacids, alpha-chloroisocaproate, phenylpyruvate, clofibric acid, octanoate and dichloroacetate. Phosphorylation of branched-chain alpha-ketoacid dehydrogenase occurs at two closely-linked serine residues (sites 1 and 2) of the alpha-subunit of the decarboxylase. HPLC and sequence data suggest homology of the amino acid sequence adjacent to phosphorylation sites 1 and 2 of complexes isolated from several different tissues. Stoichiometry for phosphorylation of all of the complexes studies was about 1 mol P/mol alpha-subunit for 95% inactivation and 1.5 mol P/mol alpha-subunit for maximally phosphorylated complex. Site 1 and site 2 were phosphorylated at similar rates until total phosphorylation exceeded 1 mol P/mol alpha-subunit. The complexes from rabbit kidney, rabbit heart, and rat heart showed 30-40% additional phosphorylation of the alpha-subunit beyond 95% inactivation. Site specificity studies carried out with the kinase partially inhibited with alpha-chloroisocaproate suggest that phosphorylation of site 1 is primarily responsible for regulation of the complex. The capacity of the branched-chain alpha-ketoacid dehydrogenase to oxidize pyruvate (Km = 0.8 mM, Vmax = 20% of that of alpha-ketoisovalerate) interferes with the estimation of activity state of the hepatic pyruvate dehydrogenase complex. The disparity between the activity states of the two complexes in most physiologic states contributes to this interference. An inhibitory antibody for branched-chain alpha-ketoacid dehydrogenase can be used to prevent interference with the pyruvate dehydrogenase assay. Almost all of the hepatic branched-chain alpha-ketoacid dehydrogenase in chow-fed rats is active (greater than 90% dephosphorylated). In contrast, almost all of the hepatic enzyme of rats fed a low-protein (8%) diet is inactive (greater than 85% phosphorylated). Fasting of chow-fed rats has no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e. greater than 90% of the enzyme remains in the active state. However, fasting of rats maintained on low-protein diets greatly activates the hepatic enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Serine/threonine protein phosphatases in the control of cell function.

Reversible protein phosphorylation is a fundamental mechanism by which many biological functions are regulated. Achievement of such control requires the coordinated action of the interconverting enzymes, the protein kinases and protein phosphatases. By comparison with protein kinases, a limited number of protein phosphatase catalytic subunits are present in the cell, which raises the question of how such a small number of dephosphorylating enzymes can counterbalance the action of the more numerous protein kinases. In mammalian cells, four major classes of Ser/Thr-specific phosphatase catalytic subunits have been identified, comprising two distinct gene families. The high degree of homology among members of the same family, PP1, PP2A and PP2B, and the high degree of evolutionary conservation between organisms as divergent as mammals and yeast, implies that these enzymes are involved in fundamental cell functions. Type 1 enzymes appear to acquire specificity by association with targeting regulatory subunits which direct the enzymes to specific cellular compartments, confer substrate specificity and control enzyme activity. In spite of the progress made in determining the structure of the PP2A subunits, very little is known about the control of this activity and about substrate selection. Recent studies have unravelled a significant number of regulatory subunits. The potential existence of five distinct B or B-related polypeptides, some of which are present in multiple isoforms, two A and two C subunit isoforms, raises the possibility that a combinatorial association could generate a large number of specific PP2A forms with different substrate specificity and/or cellular localization. Moreover, biochemical, biological and genetic studies all concur in suggesting that the regulatory subunits may play an important role in determining the properties of the Ser/Thr protein phosphatases and hence their physiological functions.

Perceptions of problems associated with aging: comparisons among four older age cohorts.

This research examines how the perceptions of aging and the concern over special problems faced by the aged vary among four age cohorts: the middle aged (55-64), the young old (65-74), the old (75-84), and the oldest old (85+). We hypothesized that the middle-aged cohort and the oldest-old cohort would be most pessimistic. The results support the hypothesis for the middle-aged group, but the oldest old were surprisingly optimistic in their view of aging. However, these perceptions by the oldest old cohort are more likely than the others' attitudes to depend on this group's assessment of their own personal problems.

In vitro and in situ inhibition of carotenoid biosynthesis in Capsicum annuum by bleaching herbicides.

Pepper leaves treated with the herbicide J852 show an accumulation of phytoene and zeta-carotene, whereas treatment with norflurazon led to an accumulation of only phytoene. The effects of these herbicides were examined in vitro after the expression of carotenoid desaturases in Escherichia coli. Whereas norflurazon is a potent inhibitor of phytoene desaturase (PDS) (I(50) = 0.12 microM) but not of zeta-carotene desaturase (ZDS) (I(50) = 144 microM), J852 inhibits both PDS (I(50) = 23 microM) and ZDS (I(50) = 49 microM). The influence of PDS/ZDS inhibition on gene expression was examined by comparative RT-PCR. None of the examined genes, namely, encoding phytoene synthase, PDS, ZDS, or the terminal oxidase associated with phytoene desaturation, were induced upon herbicide treatment in pepper leaves or seedlings. This was unexpected because inhibition of carotene desaturation led to an up-regulation of the carotenoid biosynthetic capacity (higher amounts of accumulating precursors plus remaining colored carotenoids are present in treated tissues versus control).

[Gene mapping and determination of the structure of chloroplast transfer RNA from various photosynthetic organisms]. / Kartirovanie genov i opredelenie struktury transportnykh RNK khloroplastov razlichnykh fotosinteziruiushchikh organizmov.

The review sums up data on gene mapping studies of tRNAs of chloroplasts from maize, beans, Euglena, Cyanophora. The mechanisms of splicing of tRNA2Ile from maize chloroplasts and coded for by a gene of unusual length was investigated.