Polymerization and mechanical properties of single RecA–DNA filaments

The polymerization of individual RecA-DNA filaments, containing either single-stranded or double-stranded DNA, was followed in real time, and their mechanical properties were characterized with force-measuring laser tweezers. It was found that the stretch modulus of a filament is dominated by its (central) DNA component, while its bending rigidity is controlled by its (eccentric) protein component. The longitudinal stiffness of DNA increases 6- to 12-fold when the DNA is contained in the protein helix. Both the stretch modulus and the bending rigidity of a fiber change in the presence of various nucleotide cofactors-e.g., [gamma-thio]ATP, ATP, and ADP-indicating a substantial re-arrangement of spatial relationships between the nucleic acid and the protein scaffold. In particular, when complexed with ATP, a fiber becomes twice as extensible as a [gamma-thio]ATP fiber, suggesting that 32% of the DNA-binding sites have been released in its core. Such release may enable easy rotation of the DNA within the protein helix or slippage of the DNA through the center of the protein helix.     

Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP

To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension was applied to a kinesin-microtubule rigor complex using the optical trap, and ATP was released by flash photolysis of caged ATP with a UV laser. Kinesin started to generate force and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 μM ATP, respectively. The kinetics of force generation were consistent with a two-step reaction: ATP binding, with an apparent second-order rate constant of 0.7 μM⁻¹·s⁻¹, followed by force generation at 45 s⁻¹ per kinesin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limiting step of ATPase cycle is involved with the force generation.     

Prion-inducing domain 2–114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments

The yeast non-Mendelian genetic factor [PSI], which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of [PSI]. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2–114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro. The filaments show a β-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties. It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the [PSI] element in vivo.     

Proximo–distal specification in the wing disc of Drosophila by the nubbin gene

Mutations in the nubbin (nub) gene have a phenotype consisting of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones. nub expression is restricted to the wing pouch cells in wing discs since early larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Clones located in the proximal region of the wing blade cause in addition nonautonomous reduction of the whole wing. Cell lineage experiments in a nub mutant background show that clones respect neither the anterior–posterior nor the dorsal–ventral boundary but that the selector genes have been correctly expressed since early larval development. The phenotypes of nub el and nub dpp genetic combinations are synergistic and the overexpression of dpp in clones in nub wings does not result in overproliferation of the surrounding wild-type cells. We discuss the role of nub in the wing’s proximo–distal axis and in the formation of compartment boundaries.     

Direct measurement of stiffness of single actin filaments with and without tropomyosin by in vitro nanomanipulation.

In order to explain the molecular mechanism of muscle contraction, it is crucial to know the distribution of the sarcomere compliance of active muscle. Here, we directly measure the stiffness of single actin filaments with and without tropomyosin, using a recently developed technique for nanomanipulation of single actin filaments with microneedles. The results show that the stiffness for 1-micron-long actin filaments with and without tropomyosin is 65.3 +/- 6.3 and 43.7 +/- 4.6 pN/nm, respectively. When the distribution of crossbridge forces along the actin filament is taken into account, the elongation of a 1-micron-long thin filament during development of isometric contraction is calculated to be approximately 0.23%. The time constant of force in response to a sudden length change is < 0.2 ms, indicating that the viscoelasticity is negligible in the millisecond time range. These results suggest that approximately 50% of the sarcomere compliance of active muscle is due to extensibility of the thin filaments.     

WIP, a protein associated with Wiskott–Aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells

Wiskott–Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.     

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48–CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s⁻¹. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2–CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of ≈10⁴ M⁻¹ and a dissociation rate of ≥6 s⁻¹. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.     

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF–RecO–RecR complex in DNA repair and recombination

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF–RecO–RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF–RecO–Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF–RecO–RecR complex functions as an anti-Ssb factor.     

Regulation of Bcl-xL expression in human keratinocytes by cell–substratum adhesion and the epidermal growth factor receptor

Cell–substratum adhesion is an essential requirement for survival of human neonatal keratinocytes in vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell–substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-x_L. Expression of Bcl-x_L protein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-x_L steady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-x_L expression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-x_L represents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-x_L expression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.     

Syntax processing by auditory cortical neurons in the FM–FM area of the mustached bat Pteronotus parnellii

Syntax denotes a rule system that allows one to predict the sequencing of communication signals. Despite its significance for both human speech processing and animal acoustic communication, the representation of syntactic structure in the mammalian brain has not been studied electrophysiologically at the single-unit level. In the search for a neuronal correlate for syntax, we used playback of natural and temporally destructured complex species-specific communication calls—so-called composites—while recording extracellularly from neurons in a physiologically well defined area (the FM–FM area) of the mustached bat’s auditory cortex. Even though this area is known to be involved in the processing of target distance information for echolocation, we found that units in the FM–FM area were highly responsive to composites. The finding that neuronal responses were strongly affected by manipulation in the time domain of the natural composite structure lends support to the hypothesis that syntax processing in mammals occurs at least at the level of the nonprimary auditory cortex.     

Renoprotective effect of additional sodium–glucose cotransporter 2 inhibitor therapy in type 2 diabetes patients with rapid decline and preserved renal function

Aims/IntroductionThe slope of estimated glomerular filtration rate (eGFR) decline (eGFR slope) in early‐stage type 2 diabetes patients might predict the future risk of end‐stage renal disease. Type 2 diabetes patients who show rapid progressive eGFR decline are termed rapid decliners. Several studies of rapid decliners have investigated the efficacy of sodium–glucose cotransporter 2 inhibitors (SGLT2i) in patients with advanced renal dysfunction; however, no studies, to our knowledge, have focused on patients with preserved renal function. Therefore, we investigated the efficacy of SGLT2i in rapid decliners with preserved renal function.Materials and MethodsThis study enrolled type 2 diabetes patients with baseline eGFR ≥60 mL/min/1.73 m² who had been treated with SGLT2i for ≥3 years. Among these individuals, we defined those with annual eGFR declines ≥5 mL/min/1.73 m² per year before SGLT2i administration as rapid decliners. The primary end‐point was the change in eGFR slope after SGLT2i administration.ResultsAmong 165 patients treated with SGLT2i for ≥3 years, 21 patients were rapid decliners with preserved renal function. The mean age and eGFR at SGLT2i administration were 58.6 years and 87.1 mL/min/1.73 m², respectively. The mean annual eGFR slope improved significantly in those administered SGLT2i compared with the control group (−1.00 and −4.36 mL/min/1.73 m² per year, respectively; P < 0.001). Notably, the steeper the eGFR slope before starting SGLT2i administration, the larger the improvement of eGFR slope, which was independent of the reduction of albuminuria.ConclusionsEarly intervention with SGLT2i may have renoprotective effects in type 2 diabetes patients with rapid decline and preserved renal function.     

Correction of obesity and diabetes in genetically obese mice by leptin gene therapy

The ob/ob mouse is genetically deficient in leptin and exhibits both an obese and a mild non-insulin-dependent diabetic phenotype. To test the hypothesis that correction of the obese phenotype by leptin gene therapy will lead to the spontaneous correction of the diabetic phenotype, the ob/ob mouse was treated with a recombinant adenovirus expressing the mouse leptin cDNA. Treatment resulted in dramatic reductions in both food intake and body weight, as well as the normalization of serum insulin levels and glucose tolerance. The subsequent diminishment in serum leptin levels resulted in the rapid resumption of food intake and a gradual gain of body weight, which correlated with the gradual return of hyperinsulinemia and insulin resistance. These results not only demonstrated that the obese and diabetic phenotypes in the adult ob/ob mice are corrected by leptin gene treatment but also provide confirming evidence that body weight control may be critical in the long-term management of non-insulin-dependent diabetes mellitus in obese patients.     

Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control

We have demonstrated that controlled electric fields can be used to regulate transport, concentration, hybridization, and denaturation of single- and double-stranded oligonucleotides. Discrimination among oligonucleotide hybrids with widely varying binding strengths may be attained by simple adjustment of the electric field strength. When this approach is used, electric field denaturation control allows single base pair mismatch discrimination to be carried out rapidly (<15 sec) and with high resolution. Electric field denaturation takes place at temperatures well below the melting point of the hybrids, and it may constitute a novel mechanism of DNA denaturation.     

F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF)

Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3–1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3–2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long α-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 Å, respectively, with corresponding bond angles of 99.5° and 113°, close to the optimum for a strong hydrogen bond.     

Basal expression of cyclooxygenase-2 and nuclear factor–interleukin 6 are dominant and coordinately regulated by interleukin 1 in the pancreatic islet

The enzyme cyclooxygenase (COX)-1 is constitutive whereas COX-2 is regulated in virtually all tissues. To assess whether this dogma holds true in the pancreatic islet, we examined basal and interleukin (IL)-1-regulated expression of COX-2 in HIT-T15 cells, Syrian hamster and human islets, and other Syrian hamster tissues. We found that COX-2, and not COX-1, gene expression is dominant in pancreatic islet tissue under both basal and IL-1-stimulated conditions. Control tissues (liver, spleen, and kidney) showed the expected predominance of COX-1 gene expression. Basal and IL-1-stimulated prostaglandin E₂ synthesis were blocked by a specific COX-2 inhibitor. IL-1 stimulation had a biphasic effect on COX-2 mRNA levels with an initial mild increase at 2–4 hr followed by a more dramatic decrease below basal level by 24 hr. The IL-1-induced increase in COX-2 mRNA levels was accompanied by a parallel increase in NF-κB binding to COX-2 promoter elements. The subsequent decrease in COX-2 mRNA levels was accompanied by a parallel decrease in NF-IL-6 binding activity and COX-2 promoter activity. Specific mutation of the NF-IL-6 binding motif within the COX-2 promoter reduced basal promoter activity by 50% whereas mutation of the NF-κB motif had no effect. These studies provide documentation of NF-IL-6 in the pancreatic islet and that COX-2, rather than COX-1, is dominantly expressed. They suggest coordinate regulation by IL-1 of COX-2 mRNA, NF-κB, and NF-IL-6 and raise the issue of whether intrinsically high levels of COX-2 gene expression predisposes the normal islet for microenvironmentally induced overproduction of islet prostaglandin E₂.     

Efficacy and safety of oral semaglutide in Japanese patients with type 2 diabetes: A subgroup analysis by baseline variables in the PIONEER 9 and PIONEER 10 trials

Aims/IntroductionTo assess the impact of baseline characteristics on the efficacy and safety of oral semaglutide in Japanese patients with type 2 diabetes.Materials and MethodsIn the Peptide InnOvatioN for Early diabEtes tReatment (PIONEER) 9 and 10 trials, Japanese patients were randomized to once‐daily oral semaglutide (3, 7, or 14 mg) or a comparator (placebo or once‐daily subcutaneous liraglutide 0.9 mg in PIONEER 9; once‐weekly subcutaneous dulaglutide 0.75 mg in PIONEER 10) for 52 weeks, with 5 weeks of follow up. An exploratory analysis grouped patients in each trial according to baseline glycated hemoglobin (HbA_1c; ≤8.0, >8.0–≤9.0, or >9.0%), body mass index (<25, ≥25–<30, or ≥30 kg/m²) and, for PIONEER 10 only, by background medication (sulfonylurea, glinide, thiazolidinedione, α‐glucosidase inhibitor, sodium‐glucose cotransporter 2 inhibitor). Efficacy (changes from baseline to week 26 in HbA_1c and bodyweight) and safety were assessed.ResultsSeven hundred and one patients were included (PIONEER 9: N = 243; PIONEER 10: N = 458). In both trials, HbA_1c reductions increased as baseline HbA_1c increased; there were no other apparent patterns between the variables investigated and HbA_1c or bodyweight changes. There was one statistically significant subgroup interaction between baseline HbA_1c and estimated treatment differences in bodyweight change for oral semaglutide 14 mg versus placebo in PIONEER 9 (P = 0.0286). Baseline HbA_1c, baseline body mass index and background medication did not appear to affect the proportions of patients reporting adverse events.ConclusionsOral semaglutide is effective across a range of baseline subgroups of Japanese patients with type 2 diabetes, with no unexpected safety findings.     

Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T

Head-to-tail polymerization of tropomyosin is crucial for its actin binding, function in actin filament assembly, and the regulation of actin-myosin contraction. Here, we describe the 2.1 A resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 A resolution structure of crystals containing TM-N and TM-C together with a fragment of troponin-T (TnT). At each junction, the N-terminal helices of TM-N were splayed, with only one of them packing against TM-C. In the C-terminal region of TM-C, a crucial water in the coiled-coil core broke the local 2-fold symmetry and helps generate a kink on one helix. In the presence of a TnT fragment, the asymmetry in TM-C facilitates formation of a 4-helix bundle containing two TM-C chains and one chain each of TM-N and TnT. Mutating the residues that generate the asymmetry in TM-C caused a marked decrease in the affinity of troponin for actin-tropomyosin filaments. The highly conserved region of TnT, in which most cardiomyopathy mutations reside, is crucial for interacting with tropomyosin. The structure of the ternary complex also explains why the skeletal- and cardiac-muscle specific C-terminal region is required to bind TnT and why tropomyosin homodimers bind only a single TnT. On actin filaments, the head-to-tail junction can function as a molecular swivel to accommodate irregularities in the coiled-coil path between successive tropomyosins enabling each to interact equivalently with the actin helix.     

Mutagenic effects of a single and an exact number of α particles in mammalian cells

One of the main uncertainties in risk estimation for environmental radon exposure using lung cancer data from underground miners is the extrapolation from high- to low-dose exposure where multiple traversal is extremely rare. The biological effects of a single α particle are currently unknown. Using the recently available microbeam source at the Radiological Research Accelerator Facility at Columbia University, we examined the frequencies and molecular spectrum of S1⁻ mutants induced in human–hamster hybrid (A_L) cells by either a single or an exact number of α particles. Exponentially growing cells were stained briefly with a nontoxic concentration of Hoechst dye for image analysis, and the location of individual cells was computer-monitored. The nucleus of each cell was irradiated with either 1, 2, 4, or 8 α particles at a linear energy transfer of 90 keV/μm consistent with the energy spectrum of domestic radon exposure. Although single-particle traversal was only slightly cytotoxic to A_L cells (survival fraction ≈ 0.82), it was highly mutagenic, and the induced mutant fraction averaged 110 mutants per 10⁵ survivors. In addition, both toxicity and mutant induction were dose-dependent. Multiplex PCR analysis of mutant DNA showed that the proportion of mutants with multilocus deletions increased with the number of particle traversals. These data provide direct evidence that a single α particle traversing a nucleus will have a high probability of resulting in a mutation and highlight the need for radiation protection at low doses.