cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Histone rearrangements accompany nuclear differentiation and dedifferentiation in Tetrahymena

Histone synthesis and deposition into specific classes of nuclei has been investigated in starved and conjugating Tetrahymena. During starvation and early stages of conjugation (between 0 and 5 hr after opposite mating types are mixed), micronuclei selectively lose preexisting micronuclear-specific histones α, β, γ, and H3^F. Of these histones, only α appears to accumulate in micronuclear chromatin through active synthesis and deposition during the mating process. Curiously, α is not observed (by stain or label) in young macronuclear anlagen (4C, 10 hr of conjugation). Thus, young macronuclear anlagen are missing all of the histones which are known to be specific to micronuclei of vegetative cells. By 14–16 hr of conjugation, we observe active synthesis and deposition of macronuclear-specific histones, hv1, hv2, and H1, into new macronuclear anlagen (8C). Thus macronuclear differentiation seems well underway by this time of conjugation. It is also in this time period (14–16 hr) that we first detect significant amounts of micronuclear-specific H1-like polypeptides β and γ in micronuclear extracts. These polypeptides do not seem to be synthesized during this period, which suggests that β and γ are derived from a precursor molecule(s). Since these micronuclear-specific histones do not appear in micronuclear chromatin until after other micronuclei have been selected to differentiate as macronuclei, we suspect that micronuclear differentiation is also an important process which occurs in 10–16 hr mating cells. Our results also suggest that proteolytic processing of micronuclear H3^S into H3^F (which occurs in a cell cycle dependent fashion during vegetative growth) is not operative during most if not all of conjugation. Thus micronuclei of mating cells contain only H3^S which also seems consistent with the fact that some micronuclei differentiate into new macronuclei (micronuclear H3^S is indistinguishable from macronuclear H3). Interestingly, the only H3 synthesized and deposited into the former macronucleus of mating cells is the relatively minor macronuclear-specific H3-like variant, hv2. These results demonstrate that significant histone rearrangements occur during conjugation in Tetrahymena in a manner consistent with the fact that during conjugation some micronuclei eventually differentiate into new macronuclei. Our results suggest that selective synthesis and deposition of specific histones (and histone variants) plays an important role in the nuclear differentiation process in Tetrahymena. The disappearance of specific histones also raises the possibility that developmentally regulated proteolytic processing of specific histones plays an important (and previously unsuspected) role in this system.     

Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin

The nucleolar protein gar2 of fission yeast is structurally related to the multifunctional nucleolar protein nucleolin from vertebrates and has been shown to be implicated in production of 18S rRNA. gar2 contains several potential casein kinase 2 (CK2) phosphorylation sites and a single putative p34^cdc2 phosphorylation site in the consensus S₅₀PKK. Here, we show that, like nucleolin, gar2 is phosphorylated in vitro by both highly purified CK2 from CHO cells and p34^cdc2 from starfish oocytes. Moreover, the substitution of alanine for the N-terminal serine 50 abolishes phosphorylation by p34^cdc2 in vitro. We also provide evidence that gar2 is phosphorylated in vitro by a p13^suc1-Sepharose-bound kinase from Schizosaccharomyces pombe extracts that displays cell cycle-regulated activity similar to that of the p34^cdc2 kinase. In vivo ³²P labeling of cells indicates that gar2 is a phosphoprotein and that incorporation of phosphate on residue 50 occurs specifically at mitosis. Taken together, these results lead us to propose that gar2 is likely to be an in vivo substrate for the mitotic p34^cdc2 kinase. However, this posttranslational modification of the gar2 protein does not appear to be essential for normal production of 18S rRNA. Received: 5 September 1996; in revised form: 4 February 1997 / Accepted: 24 February 1997     

Brain proline-directed protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2.

The carboxyl-terminal regions of neurofilament high (NF-H) and middle (NF-M) molecular weight proteins have been suggested to be phosphorylated in vivo by a p34cdc2-like protein kinase, on the basis of the in vivo phosphorylation site motif and in vitro phosphorylation of the proteins by p34cdc2 kinase (Hisanaga, S.I., Kusubata, M., Okumura, E. and Kishimoto, T. (1991) J. Biol. Chem. 266, 21798-21803). A novel proline-directed protein kinase previously identified and purified from bovine brain has been found in this study to phosphorylate NF-H and NF-M at sites identical to those phosphorylated by HeLa cell p34cdc2 kinase. The proline-directed kinase is composed of a 33-kDa and a 25-kDa subunit. The 33-kDa kinase subunit was partially sequenced, and degenerate oligonucleotide primers corresponding to the amino acid sequence information were used to clone the subunit by polymerase chain reaction (PCR). Two overlapping PCR products comprised a complete open reading frame of 292 amino acids. The sequence contains all features of a protein kinase, suggesting that the 33-kDa peptide represents the catalytic subunit of the kinase. The 33-kDa subunit shows high and approximately equal homology to human p34cdc2 and human cdk2, with about 58 and 59% amino acid identity, respectively. These results suggest that the brain kinase represents a new category of the cdc2 family, and that some members of the cdc2 kinase family may have major functions unrelated to cell cycle control.     

p34 Kinase-mediated release of lamins from nuclear ghosts is inhibited by cAMP-dependent protein kinase

During mitosis the lamins are found in a hyperphosphorylated and soluble state. p34^cdc2 kinase (MPF), a protein kinase complex with a pivotal role during mitosis, has been found to phosphorylate the lamins and, in some cases, though not all, to cause depolymerization of the lamina in vitro. Due to the variety of protein interactions in the lamina, there is a probable requirement for multiple enzyme activities to effect its breakdown in mitosis. Using nuclear ghosts as substrate, we have fractionated a Xenopus mitotic extract into a lamin-releasing fraction (p34^cdc2 kinase) and a fraction that inhibits p34^cdc2 kinase-mediated lamin release if the nuclear ghosts are first preincubated in it. The lamin-release-inhibiting activity in the p34^cdc2 kinase-depleted mitotic extract is, in turn, inhibited if PKI, a protein kinase inhibitor specific for PKA, is included in the preincubation reaction mixture. Furthermore, a similar degree of inhibition can be achieved by using purified PKA to preincubate the nuclear ghosts. This suggests that dephosphorylation of PKA substrate sites is necessary for lamin depolymerization.     

Regulation of actin function by protein kinase A‐mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a^−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase.     

Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15.

In fission yeast, the M-phase inducing kinase, a complex of p34cdc2 and cyclin B, is maintained in an inhibited state during interphase due to the phosphorylation of Cdc2 at Tyr15. This phosphorylation is believed to be carried out primarily by the Wee1 kinase. In human cells the negative regulation of p34cdc2/cyclin B is more complex, in that Cdc2 is phosphorylated at two inhibitory sites, Thr14 and Tyr15. The identities of the kinases that phosphorylate these sites are unknown. Since fission yeast Wee1 kinase behaves as a dual-specificity kinase in vitro, a popular hypothesis is that a human Wee1 homolog might phosphorylate p34cdc2 at both sites. We report here that a human gene, identified as a possible Wee1 homologue, blocks cell division when overexpressed in HeLa cells. This demonstrates functional conservation of the Wee1 mitotic inhibitor. Contrary to the dual-specificity kinase hypothesis, purified human Wee1 phosphorylates p34cdc2 exclusively on Tyr15 in vitro; no Thr14 phosphorylation was detected. Human and fission yeast Wee1 also specifically phosphorylate synthetic peptides at sites equivalent to Tyr15. Mutation of a critical lysine codon (Lys114) believed to be essential for kinase activity abolished both the in vivo mitotic inhibitor function and in vitro kinase activities of human Wee1. These results conclusively prove that Wee1 kinases inhibit mitosis by directly phosphorylating p34cdc2 on Tyr15, and strongly indicate that human cells have independent kinase pathways directing the two inhibitor phosphorylations of p34cdc2.     

In isolated human centrosomes,the associated kinases phosphorylate a specific subset of centrosomal proteins

Summary— Several studies have shown that kinases and phosphatases can interact with the centrosome during interphase and mitosis suggesting that centrosomal components might be the targets of these enzymes. The association of the cAMP-dependent protein kinase type II and the mitotic kinase p34^cdc2 with centrosomes from human lymphoblast cells has previously been shown (Keryer et al, 1993, Exp Cell Res 204, 230–240; Bailly et al, 1989, EMBO J 8, 3985–3995). In this paper we demonstrate that isolated centrosomes are able to phosphorylate a few number of centrosomal proteins (M_r 230–220000; 135000 and 50000) and also H1 histone. The phosphorylation of H1-histone is cell cycle dependent and modulated by phosphatases. The use of kinase and phosphatase inhibitors and the addition of the catalytic subunit of cAMP-dependent kinase or of cyclinB-p34^cdc2 kinase showed that both kinases phosphorylate the same centrosomal substrates. In addition two centrosomal proteins (M_r 100000 and 37000) were phosphorylated only by p34^cdc2 kinase. Although the low amount of centrosomal proteins precluded a full characterization of these substrates we discuss the identity of the major centrosomal phosphoproteins by comparison with proteins known to associate with microtubule-organizing centres or mitotic spindles. Our results raise also the intriguing possibility that the cAMP-dependent protein kinase could be regulated by the mitotic kinase at the entry of mitosis.     

Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells

The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c).     

Regulation of maturation-promoting factor by protein kinase C in Chaetopterus oocytes

Summary

We present the results of a variety of studies showing that activation of protein kinase C (PKC) in oocytes of Chaetopterus pergamentaceus results in germinal vesicle breakdown (GVBD). Phorbol esters and diacylglycerol can initiate a morphologically normal GVBD accompanied by a spectrum of associated biochemical processes, including increased protein phosphorylation, a shift in protein synthesis and activation of a protein kinase, maturation promoting factor (MPF). MPF activation is essential for GVBD in response to phorbol esters. In addition, inhibitors of PKC can block naturally-induced GVBD. We also present evidence that PKC can phosphorylate p34^cde2, the catalytic subunit of MPF and that phosphorylation by PKC increases the histone H1 kinase activity of immunoprecipitated MPF. Immunoblot studies show that Chaetopterus oocyte p34^cdc2 is not tyrosine phosphorylated prior to the initiation of GVBD, indicating that activation of MPF at GVBD in this species does not require p80^cdc25, the activator of MPF at mitosis. These results suggest that PKC is an essential regulator of GVBD which can directly phosphorylate and regulate p34^cdc2. Since PKC is the intracellular receptor for and is directly activated by tumor-promoters, tumor promotion might involve acceleration of the cell cycle through modification of the enzymatic activity of MPF by PKC.     

Studies on the reaction specificity of the flavoprotein lysine monooxygenase with modified substrates

Various modified substrates of lysine monooxygenase were examined to determine whether they were oxygenated or oxidized. Among various methyllysines tested, N^?- and δ-methyllysine underwent predominantly an oxygenative decarboxylation, producing the corresponding acid amides, while γ-methyllysine underwent predominantly an oxidative deamination with an α-keto acid as the reaction product. β-Methyllysine was inactive as substrate. All four methyllysines decreased the cooperativity of the enzyme with the normal substrate, lysine. Furthermore, lysine oxygenation was competitively inhibited by all of them except for β-methyllysine, which was much less inhibitory than the other methyllysines. Other analogs with a chloro or hydroxyl group at either the δ or the γ position were both oxygenated and oxidized. Analogs with a modified carboxyl or α-amino group were inactive as substrates.     

Casein kinase 1δ activates human recombinant deoxycytidine kinase by Ser-74 phosphorylation, but is not involved in the in vivo regulation of its activity

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI δ. We showed that recombinant CKI δ phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI δ correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI δ, strengthening the key role of this residue in the control of dCK activity. However, neither CKI δ inhibitors nor CKI δ siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI δ in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI δ could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.     

Mitogen-activated protein kinases phosphorylate nuclear lamins and display sequence specificity overlapping that of mitotic protein kinase p34cdc2.

Members of the mitogen-activated protein (MAP) kinase family are implicated in mediating entry of cells into the cell cycle, as well as passage through meiotic M phase. These kinases have attracted much interest because their activation involves phosphorylation on both tyrosine and threonine residues, but little is known about their physiological targets. In this study, two distinct members of the MAP kinase family (p44mpk and p42mapk) are shown to phosphorylate chicken lamin B2 at a single site identified as Ser16. Moreover, these MAP kinases cause depolymerization of in-vitro-assembled longitudinal lamin head-to-tail polymers. Ser16 was previously shown to be phosphorylated during mitosis in vivo, and to be a target of the mitotic protein kinase p34cdc2 in vitro. Accordingly, lamins were proposed to be direct in vivo substrates of p34cdc2. This proposal is supported by quantitative analyses indicating that lamin B2, when assayed in vitro, is a substantially better substrate for p34cdc2 than for MAP kinases. Nevertheless, a physiological role of MAP kinases in lamin phosphorylation is not excluded. The observation that members of the MAP kinase family display sequence specificities overlapping that of p34cdc2 raises the possibility that some of the purported substrates of p34cdc2 may actually be physiological substrates of MAP kinases.     

cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication.

RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.     

Antioxidant Effect of Tocopherols on Autoxidation of Milk Fat

Antioxidant activity of d-α-, dl-β-, d-γ- and d-δ-tocopherol was investigated with fatty acid methylester of milk fat from which unsaponifiable matter had been removed. Autoxidation was carried out at 50°C and its degree was indicated by peroxide value, α- or β-Tocopherol was more effective at lower concentrations (0.003 and 0.01%) than at higher concentrations (0.05, 0.1 and 0.5%). The antioxidant activity of γ- and δ-tocopherol was increased with the increase of tocopherol concentration within the range of 0.001 to 0.5%. The order of antioxidant activity of these tocopherols, which was compared in terms of the time to reach 30 meq of peroxide value, varied with the concentration; γ > β > δ > α at 0.001%, α > γ > β > δ at 0.003%, γ > δ > β > α at 0.01%, and δ > γ > β > α at the concentrations more than 0.05%. α-Tocopherol at the concentration of 0.003%, which corresponded to the concentration in original milk fat, was more effective than other tocopherols at the same concentration and α-tocopherol at other concentrations. Synergism due to the combination of β-, γ-, or δ-tocopherol with 0.003% of α-tocopherol was not observed.     

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10^?5m epinephrine or norepinephrine resulted in a rapid incorporation of ³²P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, ³²P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each ³²P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each ³²P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain ³²P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ-³²P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with ³²P_i resulted in phosphorylation of the same amino acid residues.     

Phosphorylation of neuronal nitric oxide synthase at Ser in the dentate gyrus of rat brain after transient forebrain ischemia

We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser⁸⁴⁷ in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser¹⁴¹²in vitro. In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser¹⁴¹² after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24 h, after 15 min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated (p)-nNOS at Ser¹⁴¹², PKA, and p-PKA at Thr¹⁹⁷ were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p-nNOS at Ser¹⁴¹² significantly increased between 1 and 6 h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser¹⁴¹² and PKA at Thr¹⁹⁷ occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr¹⁹⁷, which then phosphorylates nNOS at Ser¹⁴¹² in the subgranular layer of the dentate gyrus.     

Definitive expression of c-mos in late meiotic prophase leads to phosphorylation of a 34 kda protein in cultured rat spermatocytes

To investigate the role of c-mos in rat spermatogenesis, expression of c-mos, MAP kinase kinase (MAPKK), MAP kinase (MAPK), cdc2 and protein kinase A (PKA) by spermatogenic cell culture of 14 day-old rats was examined. MAPKK and PKA expressions were constitutive, whereas the expression of MAPK and cdc2 in spermatogonia initially decreased, but later increased on meiotic maturation of spermatocytes. c-mos expression was definitive of late meiotic prophase. c-mos immunoprecipitates prepared from the c-mos-enriched fraction (pI9.0-9.6) could form complex(es) in the cultured spermatogenic cell lysates. In vitro phosphorylation of the c-mos immune complexes revealed a 34 kDa protein that was phosphorylated at serine and threonine residues as a target of the c-mos signal. Its pI value was 4.4-4.5, and cdc2 was not detected, making it different from cdc2 (p34). These results suggest that the phosphorylation of the 34 kDa protein by the c-mos signal may play a crucial role in the meiotic division of rat spermatocytes.     

The Enantioselectivity of the Cellular Deoxynucleoside Kinases

Abstract

Cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK), phosphorylate deoxynucleosides and their analogs. Recombinant human TK1 only phosphorylated β-D Thd, but recombinant TK2, dCK and dGK all phosphorylated equally well β-D and β-L as well as to some extent α-D and α-L deoxynucleosides.