In silico structure-based discovery of a SARS-CoV-2 main protease inhibitor

The Coronavirus Disease 2019 (COVID-19) pandemic caused by the novel lineage B betacoroanvirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant mortality, morbidity, and socioeconomic disruptions worldwide. Effective antivirals are urgently needed for COVID-19. The main protease (M^pro) of SARS-CoV-2 is an attractive antiviral target because of its essential role in the cleavage of the viral polypeptide. In this study, we performed an in silico structure-based screening of a large chemical library to identify potential SARS-CoV-2 M^pro inhibitors. Among 8,820 compounds in the library, our screening identified trichostatin A, a histone deacetylase inhibitor and an antifungal compound, as an inhibitor of SARS-CoV-2 M^pro activity and replication. The half maximal effective concentration of trichostatin A against SARS-CoV-2 replication was 1.5 to 2.7µM, which was markedly below its 50% effective cytotoxic concentration (75.7µM) and peak serum concentration (132µM). Further drug compound optimization to develop more stable analogues with longer half-lives should be performed. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of SARS-CoV-2.     

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro

COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CL^pro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CL^pro activity in vitro with IC₅₀’s of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC₅₀’s of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CL^pro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CL^pro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.     

Structural and kinetic analyses of holothurian sulfated glycans suggest potential treatment for SARS-CoV-2 infection

Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10–60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-β-GalNAcX-(1→4)-β-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO₃⁻. The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin. IC₅₀ values demonstrated the activity of the three holothurian sulfated glycans to be ∼12 times more efficient than heparin, with no cytotoxic effects. The dissociation constant (K_D) values obtained by surface plasmon resonance of the wildtype SARS-CoV-2 spike (S)-protein receptor-binding domain (RBD) and N501Y mutant RBD in interactions with the heparin-immobilized sensor chip were 94 and 1.8 × 10³ nM, respectively. Competitive surface plasmon resonance inhibition analysis of PpFucCS, IbFucCS, and IbSF against heparin binding to wildtype S-protein showed IC₅₀ values (in the nanomolar range) 6, 25, and 6 times more efficient than heparin, respectively. Data from computational simulations suggest an influence of the sulfation patterns of the Fuc units on hydrogen bonding with GlcA and that conformational change of some of the oligosaccharide structures occurs upon S-protein RBD binding. Compared with heparin, negligible anticoagulant action was observed for IbSF. Our results suggest that IbSF may represent a promising molecule for future investigations against SARS-CoV-2.     

Simple Uniaxial and Uniform Biaxial Deformation of Nearly Isotropic Incompressible Tissues

A method is developed for analyzing in a unified manner both uniaxial and uniform biaxial strain data obtained from nearly isotropic tissues. The formulation is a direct application of nonlinear elasticity theory pertaining to large deformations. The general relation between Eulerian stress (σ) and extension ratio (λ) in soft isotropic elastic bodies undergoing uniform deformation takes the simple form: σ = ((λ³ - 1)/λ) f(λ), where f(λ) must be determined for each material. The extension ratio may be either greater than 1.0 (uniaxial elongation), or lie between zero and 1.0 (uniform biaxial extension). Simple analytical functions for f(λ) are most readily found for each tissue by plotting all data as (λ³ - 1)/λσ vs. λ. Of those tissues investigated in this way (dog pericardium and pleura, and cat mesentery and dura), all but pleura could be adequately described by a parabola: 1/f(λ) = 1/k{[(λ_M - λ)(λ - λ_m)]/[λ_M - λ_m}. In these instances, three material constants per tissue (K, λ_M, λ_m) served to predict approximately the stresses attained during both small and large deformations, in strips and sheets alike. It was further found that the uniaxial strain asymptote (λ_M) was linearly related to the biaxial strain asymptote (Λ_M), thus effectively reducing the number of constants by one.     

Diffusion Tensor Magnetic Resonance Imaging of the Pancreas

Purpose

To develop a diffusion-tensor-imaging (DTI) protocol that is sensitive to the complex diffusion and perfusion properties of the healthy and malignant pancreas tissues.

Materials and Methods

Twenty-eight healthy volunteers and nine patients with pancreatic-ductal-adenocacinoma (PDAC), were scanned at 3T with T2-weighted and DTI sequences. Healthy volunteers were also scanned with multi-b diffusion-weighted-imaging (DWI), whereas a standard clinical protocol complemented the PDAC patients’ scans. Image processing at pixel resolution yielded parametric maps of three directional diffusion coefficients λ1, λ2, λ3, apparent diffusion coefficient (ADC), and fractional anisotropy (FA), as well as a λ1-vector map, and a main diffusion-direction map.

Results

DTI measurements of healthy pancreatic tissue at b-values 0,500 s/mm²yielded: λ1 = (2.65±0.35)×10⁻³, λ2 = (1.87±0.22)×10⁻³, λ3 = (1.20±0.18)×10⁻³, ADC = (1.91±0.22)×10⁻³ (all in mm²/s units) and FA = 0.38±0.06. Using b-values of 100,500 s/mm² led to a significant reduction in λ1, λ2, λ3 and ADC (p<.0001) and a significant increase (p<0.0001) in FA. The reduction in the diffusion coefficients suggested a contribution of a fast intra-voxel-incoherent-motion (IVIM) component at b≤100 s/mm², which was confirmed by the multi-b DWI results. In PDACs, λ1, λ2, λ3 and ADC in both 0,500 s/mm² and 100,500 s/mm² b-values sets, as well as the reduction in these diffusion coefficients between the two sets, were significantly lower in comparison to the distal normal pancreatic tissue, suggesting higher cellularity and diminution of the fast-IVIM component in the cancer tissue.

Conclusion

DTI using two reference b-values 0 and 100 s/mm² enabled characterization of the water diffusion and anisotropy of the healthy pancreas, taking into account a contribution of IVIM. The reduction in the diffusion coefficients of PDAC, as compared to normal pancreatic tissue, and the smaller change in these coefficients in PDAC when the reference b-value was modified from 0 to 100 s/mm², helped identifying the presence of malignancy.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Bacteriorhodopsin wildtype and variant aspartate-96 → aspargine as reversible holographic media

Air dried films of purple membranes (PM) from Halobacterium halobium containing the photochromic protein bacteriorhodopsin (BR) were prepared and the BR-photocycle of this material analyzed. The absorption maxima of the initial state B (λ_max = 570 nm) and the photochemical intermediate M (λ_max = 412 nm), which is the longest living intermediate in suspension (τ ≈ 10 ms), were spectrally well separated. Light-induced population gratings between B and M were used for reversible holographic recording in these dry PM films. The resolution (>5,000 lines/mm) of PM films was comparable to the corresponding values of conventional photochromic recording materials. The longterm stability toward photochemical degradation of PM films is excellent (> 100.000 recording cycles). The spectral bandwidth (400-680 nm) of such films covers nearly the whole visible spectrum. Both the photochemical transition from B → M with wavelengths in the green-red range and from M → B with blue light were utilized for holographic recording. The latter possibility (M → B) seems to be advantageous for several applications because the holographic grating is only formed during reconstruction. Higher reading intensities lead to higher population of the M-state and result in an increase of the fringe contrast instead of decreasing it. New possibilities for the further development of holographic media based on bacteriorhodopsin are raised by the availability of PM variants with modified optical properties. By the use of the variant BR-326, which differs from the wildtype PM by a single amino acid exchange (aspartate-96 → asparagine), the sensitivity of PM films is increased by ~50% from 12 cm²/J to 19 cm²/J for recording with 568 nm. The sensitivity for recording with 413 nm (33 cm²/J) is not influenced by the amino acid exchange. The observed diffraction efficiency η of PM films with BR-326 is twice that of BR-wildtype (BR-WT) films and is in the range of conventional organic photochromics (≈ 1%). In dried films of both BR-WT and BR-326 the M-decay was shown to be at least biexponential.     

Molecular Mechanism of Heat Shock-Provoked Disassembly of the Coliphage λ Replication Complex

We have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43°C resulted in the decay of the λO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of λ plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from λ DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroEL/S and ClpP/ClpX proteins. In contrast to λcro⁺, in λcro⁻ plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the λcrots plasmid-harboring cells, a Cro repressor-independent control of λ DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to λ DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the λcro⁻ plasmid replication control.     

Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (M^pro, also called 3CL^pro) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here, we report the biophysical and enzymatic characterization of native M^pro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high-throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate, which is better suited for high-throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus M^pro enzyme assays to facilitate the discovery and development of therapies targeting M^pro.     

Functional dynamics of SARS-CoV-2 3C-like protease as a member of clan PA

SARS-CoV-2 3C-like protease (3CL^pro), a potential therapeutic target for COVID-19, consists of a chymotrypsin fold and a C-terminal α-helical domain (domain III), the latter of which mediates dimerization required for catalytic activation. To gain further understanding of the functional dynamics of SARS-CoV-2 3CL^pro, this review extends the scope to the comparative study of many crystal structures of proteases having the chymotrypsin fold (clan PA of the MEROPS database). First, the close correspondence between the zymogen-enzyme transformation in chymotrypsin and the allosteric dimerization activation in SARS-CoV-2 3CL^pro is illustrated. Then, it is shown that the 3C-like proteases of family Coronaviridae (the protease family C30), which are closely related to SARS-CoV-2 3CL^pro, have the same homodimeric structure and common activation mechanism via domain III mediated dimerization. The survey extended to order Nidovirales reveals that all 3C-like proteases belonging to Nidovirales have domain III, but with various chain lengths, and 3CL^pro of family Mesoniviridae (family C107) has the same homodimeric structure as that of C30, even though they have no sequence similarity. As a reference, monomeric 3C proteases belonging to the more distant family Picornaviridae (family C3) lacking domain III are compared with C30, and it is shown that the 3C proteases are rigid enough to maintain their structures in the active state.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12551-022-01020-x.     

Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy

The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3C^pro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3C^pro, we measured cleavage kinetics of predicted SVV-001 3C^pro substrates. An efficient substrate, L/VP4 (k_cat/K_M = 1932 ± 183 M^-1s^-1), was further optimized by a P2’ N→P substitution yielding L/VP4.1 (k_cat/K_M = 17446 ± 2203 M^-1s^-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3C^pro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3C^pro, with direct implications for anti-cancer therapeutic development.     

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M^pro) and papain like protease (PL^pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851M^pro inhibitors and 205 PL^pro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M^pro and PL^pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M^pro inhibitors and over 20% of PL^pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M^pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.     

Cross-variant protection against SARS-CoV-2 infection in hamsters immunized with monovalent and bivalent inactivated vaccines

Rapid development and successful use of vaccines against SARS-CoV-2 might hold the key to curb the ongoing pandemic of COVID-19. Emergence of vaccine-evasive SARS-CoV-2 variants of concern (VOCs) has posed a new challenge to vaccine design and development. One urgent need is to determine what types of variant-specific and bivalent vaccines should be developed. Here, we compared homotypic and heterotypic protection against SARS-CoV-2 infection of hamsters with monovalent and bivalent whole-virion inactivated vaccines derived from representative VOCs. In addition to the ancestral SARS-CoV-2 Wuhan strain, Delta (B.1.617.2; δ) and Theta (P.3; θ) variants were used in vaccine preparation. Additional VOCs including Omicron (B.1.1.529) and Alpha (B.1.1.7) variants were employed in the challenge experiment. Consistent with previous findings, Omicron variant exhibited the highest degree of immune evasion, rendering all different forms of inactivated vaccines substantially less efficacious. Notably, monovalent and bivalent Delta variant-specific inactivated vaccines provided optimal protection against challenge with Delta variant. Yet, some cross-variant protection against Omicron and Alpha variants was seen with all monovalent and bivalent inactivated vaccines tested. Taken together, our findings support the notion that an optimal next-generation inactivated vaccine against SARS-CoV-2 should contain the predominant VOC in circulation. Further investigations are underway to test whether a bivalent vaccine for Delta and Omicron variants can serve this purpose.     

Two novel human and mouse DNA polymerases of the polX family

We describe here two novel mouse and human DNA polymerases: one (pol λ) has homology with DNA polymerase β while the other one (pol µ) is closer to terminal deoxynucleotidyltransferase. However both have DNA polymerase activity in vitro and share similar structural organization, including a BRCT domain, helix–loop–helix DNA-binding motifs and polymerase X domain. mRNA expression of pol λ is highest in testis and fetal liver, while expression of pol µ is more lymphoid, with highest expression both in thymus and tonsillar B cells. An unusually large number of splice variants is observed for the pol µ gene, most of which affect the polymerase domain. Expression of mRNA of both polymerases is down-regulated upon treatment by DNA damaging agents (UV light, γ-rays or H₂O₂). This suggests that their biological function may differ from DNA translesion synthesis, for which several DNA polymerase activities have been recently described. Possible functions are discussed.     

Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL^pro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL^pro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL^pro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL^pro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL^pro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL^pro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL^pro domain was found to suppress IFN-β promoter activation, PL^pro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL^pro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PL^pro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL^pro as a viral DUB during MERS-CoV infection.     

Chemical role of α-tocopherol in salt stress mitigation by improvement in morpho-physiological attributes of sunflower (Helianthus annuus L.)

Elevated concentrations of salts in soil and water represent abiotic stresses. It considerably restricts plant productivity. However, the use of alpha-tocopherol (α-toc) as foliar can overcome this problem. It can improve crop productivity grown under salinity stress. Limited literature is documented regarding its optimum foliar application on sunflower. That’s why the need for the time is to optimize α-toc foliar application rates for sunflower cultivated in salt-affected soil. A pot experiment was performed to select a better α-toc foliar application for mitigation of salt stress in different sunflower cultivars FH (572 and 621). There were 2 levels of salts, i.e., control (no salt stress) and sodium chloride (120 mM) and four α-toc foliar application (0, 100, 200, and 300 mg L⁻¹). Results showed that foliar application of 100 mg/L- α-toc triggered the remarkable increase in fresh shoot weight, fresh root weight, shoot, and root lengths under salinity stress in FH-572 and FH-621 over 0 mg/L- α-toc. Foliar application of 200 mg/L- α-toc was most effective for improvement in chlorophyll a, chlorophyll b, total chlorophyll and carotenoids compared to 0 mg/L- α-toc. Furthermore, an increase in A was noted in FH-572 (17%) and FH-621 (22%) with α-toc (300 mg L⁻¹) application under saline condition. In conclusion, the 100 and 200 mg/L- α-toc are the best application rates for the improvement in sunflower FH-572 and FH-621 growth, chlorophyll contents and gas exchange attributes. Further investigations are needed to select a better foliar application rate between 100 and 200 mg/L- α-toc at the field level under the different agro-climatic zone and soil types.     

THE EFFECT OF UNILATERAL ULTRAVIOLET LIGHT ON THE DEVELOPMENT OF THE FUCUS EGG

1. When Fucus eggs which have been fertilized for a sufficient length of time are irradiated unilaterally with monochromatic ultraviolet light (λ2804 Å) of adequate dosage, 97–100 per cent form rhizoids on the halves of the eggs away from the source of radiation (see Figs. 1 and 2). 2. The responsiveness of the eggs increases gradually after fertilization and does not reach a maximum until about 7 hours at 15°C. (see Fig. 3). The first rhizoids begin to form in a population at about 12 hours after fertilization. The responsiveness remains maximal until at least 11 hours after fertilization. 3. It is suggested that the low responsiveness of a population of eggs at an earlier period is due to recovery from the effects of irradiation before the rhizoids begin to form. 4. The response of eggs to λ2804 Å is proportional, over a wide range, to the logarithm of the dosage (see Fig. 1). Dosage was regulated by the duration of exposure during the period of maximum response. 5. High dosages of λ2804 Å, of the order of 10,000 ergs per mm.², cause the rhizoids to form fairly precisely away from the source of radiation (see Fig. 2). Twice this dosage inhibits rhizoid formation altogether without causing cytolysis. 6. Other wave-lengths which have also been shown to be effective are: 3660, 3130, 2654, 2537, 2482, and 2345 Å. Only exploratory measurements have been made to test the effectiveness of these wave-lengths, but they show that much greater energy is necessary to obtain a strong response with λ3130 and 3660 Å, especially the latter. The wave-lengths shorter than 2804 Å, on the other hand, show the same order of effectiveness as λ2804 Å. Some may be more effective. 7. A beam of λ2804 Å which is incident on a single layer of Fucus eggs is completely extinguished at 2, 3, 6, or 6½ hours after fertilization. About 85 per cent of a beam of λ3660 Å is extinguished. The wave-length 3660 Å is thus not so completely absorbed as λ2804 Å, but the difference in proportion absorbed by the egg is not nearly so great as the difference in effectiveness.     

A specific N-terminal extension of the 8 kDa domain is required for DNA end-bridging by human Polμ and Polλ

Human DNA polymerases mu (Polµ) and lambda (Polλ) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3′ single-stranded overhangs and trans-polymerization using one 3′ end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence (‘brooch’), located at the N-terminal boundary of the Polß-like polymerase core, and formed by Tyr¹⁴¹, Ala¹⁴², Cys¹⁴³, Gln¹⁴⁴ and Arg¹⁴⁵ in Polµ, and by Trp²³⁹, Val²⁴⁰, Cys²⁴¹, Ala²⁴² and Gln²⁴³ in Polλ. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Polµ. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro.     

State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F₀ level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (τ ≈ 85 ps, λ_max^em ≈ 695-700 nm), open PS II α-centers (τ ≈ 300 ps, λ_max^em = 685 nm), and open PS II β-centers (τ ≈ 600 ps, λ_max^em = 685 nm). A fourth component of very low amplitude (τ ≈ 2.2-2.3 ns, λ_max^em = 685 nm) derives from dead chlorophyll. (b) At the F_max level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F₀ level, from closed PS II α-centers (τ ≈ 2.2 ns, λ_max^em = 685 nm) and from closed PS β-centers (τ ≈ 1.2 ns, λ_max^em = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of α- and β-units brought about by the reversible migration of light-harvesting complexes between α-centers and β-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II α,β-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202).