Characterization of poly(ADP-ribosyl)ated domains of rat pachytene chromatin

Poly(ADP-ribosyl)ation of nuclear proteins was several-fold higher in the pachytene spermatocytes than in the premeiotic germ cells of the rat. Among the histones of the pachytene nucleus, histone subtypes H2A, H1 and H3 were poly(ADP-ribosyl)ated. Based on the immunoaffinity fractionation procedure of Malik, Miwa, Sugimara & Smulson [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] we have fractionated DNAase-II-solubilized chromatin into poly(ADP-ribosyl)ated chromatin (PAC) and non-poly(ADP-ribosyl)ated chromatin (non-PAC) domains on an anti-[poly(ADP-ribose)] IgG affinity matrix. Approx. 2.5% of the pachytene chromatin represented the PAC domains. A significant amount of [alpha-32P]dATP-labelled pachytene chromatin (labelled in vitro) was bound to the affinity matrix. The DNA of pachytene PAC domains had internal strand breaks, significant length of gaps and ligatable ends, namely 5'-phosphoryl and 3'-hydroxyl termini. On the other hand, the PAC domains from 18 h regenerating liver had very few gaps, if any. The presence of gaps in the pachytene PAC DNA was also evident from thermal denaturation studies. Although many of the polypeptides were common to the PAC domains of both pachytene and regenerating liver, the DNA sequences associated with these domains were quite different. A 20 kDa protein and the testis-specific histone H1t were selectively enriched in the pachytene PAC domains. The pachytene PAC domains also contained approx. 10% of the messenger coding sequences present in the DNAase-II-solubilized chromatin. The pachytene PAC domains, therefore, may represent highly enriched DNA-repair domains of the pachytene nucleus.

Mixed-mode stress intensity factors for arbitrarily oriented cracks in cylindrical shells under axial pull and torsion

Results of photoelastic investigations conducted on cylindrical tubes (made of Araldite material) containing cracks oriented at 0°, 30°, 45°, 60° and 90° to the axis of the tube and subjected to axial and torsional loads are reported. The stress-intensity factors (SIFs) were determined by analysing the crack-tip stress fields. Smith and Smith's method [Engng Fracture Mech.4, 357–366 (1972)] and a new method developed by the authors by modifying Rakesh et al.'s method [Proc. 26th Congress of ISTAM, India (1981)] were employed to evaluate the mixed-mode SIFs.

Regulation of cytochrome P-450b/e gene expression by a heme- and phenobarbitone-modulated transcription factor

The cloned DNA fragment of the cytochrome P-450b/e gene containing the upstream region from position -179 through part of the first exon is faithfully transcribed in freeze-thawed rat liver nuclei. Phenobarbitone treatment of the animal strikingly increases this transcription, and the increase is blocked by cycloheximide (protein synthesis inhibitor) or CoCl2 (heme biosynthetic inhibitor) treatment of animals. This picture correlates very well with the reported cytochrome P-450b/e mRNA levels in vivo and run-on transcription rates in vitro under these conditions. The upstream region (from position -179) was assessed for protein binding with nuclear extracts by nitrocellulose filter binding, gel retardation, DNase I treatment ("footprinting"), and Western blot analysis. Phenobarbitone treatment dramatically increases protein binding to the upstream region, an increase once again blocked by cycloheximide or CoCl2 treatments. Addition of heme in vitro to heme-deficient nuclei and nuclear extracts restores the induced levels of transcription and protein binding to the upstream fragment, respectively. Thus, drug-mediated synthesis and heme-modulated binding of a transcription factor(s) appear involved in the transcriptional activation of the cytochrome P-450b/e genes, and an 85-kDa protein may be a major factor in this regard.

Supramolecular insulin assembly II for a sustained treatment of type 1 diabetes mellitus

Diabetes is a chronic disease requiring continuous medical supervision and patient education to prevent acute secondary complications. In this study, we have harnessed the inherent property of insulin to aggregate into an oligomeric intermediate on the pathway to amyloid formation, to generate a form that exhibits controlled and sustained release for extended periods. Administration of a single dose of the insulin oligomer, defined here as the supramolecular insulin assembly II (SIA-II), to experimental animals rendered diabetic by streptozotocin or alloxan, released the hormone capable of maintaining physiologic glucose levels for > 120 days for bovine and > 140 days for recombinant human insulin without fasting hypoglycemia. Moreover, the novel SIA-II described here not only improved the glycemic control, but also reduced the extent of secondary diabetic complications.

Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge

Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.

Some implications of an alternative structure for DNA

We have constructed a space-filling (Corey-Pauling-Koltun) model of an alternative structure for DNA. This structure is not a double helix, but consists of a pair of polynucleotide strands lying side by side and held together by Watson-Crick base pairing. Each of the two strands has alternating right- and left-handed helical segments approximately five base pairs in length. Sugar residues in alternating segments along a strand point in opposite directions. A structure slightly different from the present one proposed earlier by ourselves and another group and in which sugars in a strand all point in the same direction is ruled out. The present structure yields natural solutions to the problems of supercoiling of DNA and of strand separation during DNA replication. This model is energetically more favorable than the double helix.

Distributed algorithms for edge dominating sets

An edge dominating set for a graph G is a set D of edges such that each edge of G is in D or adjacent to at least one edge in D. This work studies deterministic distributed approximation algorithms for finding minimum-size edge dominating sets. The focus is on anonymous port-numbered networks: there are no unique identifiers, but a node of degree d can refer to its neighbours by integers 1, 2, ..., d. The present work shows that in the port-numbering model, edge dominating sets can be approximated as follows: in d-regular graphs, to within 4 − 6/(d + 1) for an odd d and to within 4 − 2/d for an even d; and in graphs with maximum degree Δ, to within 4 − 2/(Δ − 1) for an odd Δ and to within 4 − 2/Δ for an even Δ. These approximation ratios are tight for all values of d and Δ: there are matching lower bounds.

Fast distributed approximation algorithms for vertex cover and set cover in anonymous networks

We present a distributed algorithm that finds a maximal edge packing in O(Δ + log* W) synchronous communication rounds in a weighted graph, independent of the number of nodes in the network; here Δ is the maximum degree of the graph and W is the maximum weight. As a direct application, we have a distributed 2-approximation algorithm for minimum-weight vertex cover, with the same running time. We also show how to find an f-approximation of minimum-weight set cover in O(f2k2 + fk log* W) rounds; here k is the maximum size of a subset in the set cover instance, f is the maximum frequency of an element, and W is the maximum weight of a subset. The algorithms are deterministic, and they can be applied in anonymous networks.

An optimal local approximation algorithm for max-min linear programs

In a max-min LP, the objective is to maximise ω subject to Ax ≤ 1, Cx ≥ ω1, and x ≥ 0 for nonnegative matrices A and C. We present a local algorithm (constant-time distributed algorithm) for approximating max-min LPs. The approximation ratio of our algorithm is the best possible for any local algorithm; there is a matching unconditional lower bound.

Estimation Of Charge Transport Parameters And Equivalent Circuit For Poly Alkyl Thiophene Field-Effect Transistors

The small signal ac response is measured across the source-drain terminals of organic field-effect transistors (OFET) under dc bias to obtain the equivalent circuit parameters of poly(2,5-bis(3-tetradecylthiophen-2-yl)thieno[3,2-b]thiophene) (PBTTT) and poly(3-hexyl thiophene) (P3HT) based devices. The numerically simulated response based on these parameters is in good agreement with the experimental data for PBTTT-FET except at low frequencies, while the P3HT-FET data show significant deviations. This indicates that the interface with the metal electrode is rather complex for the latter, involving additional circuit elements arising from contact impedance or charge injection processes. Such an investigation can help in identifying the operational bottlenecks and to improve the performance of OFETs.

Locally checkable proofs

This work studies decision problems from the perspective of nondeterministic distributed algorithms. For a yes-instance there must exist a proof that can be verified with a distributed algorithm: all nodes must accept a valid proof, and at least one node must reject an invalid proof. We focus on locally checkable proofs that can be verified with a constant-time distributed algorithm. For example, it is easy to prove that a graph is bipartite: the locally checkable proof gives a 2-colouring of the graph, which only takes 1 bit per node. However, it is more difficult to prove that a graph is not bipartite—it turns out that any locally checkable proof requires Ω(log n) bits per node. In this work we classify graph problems according to their local proof complexity, i.e., how many bits per node are needed in a locally checkable proof. We establish tight or near-tight results for classical graph properties such as the chromatic number. We show that the proof complexities form a natural hierarchy of complexity classes: for many classical graph problems, the proof complexity is either 0, Θ(1), Θ(log n), or poly(n) bits per node. Among the most difficult graph properties are symmetric graphs, which require Ω(n2) bits per node, and non-3-colourable graphs, which require Ω(n2/log n) bits per node—any pure graph property admits a trivial proof of size O(n2).

The evolution of genomic imprinting

We explore three possible pathways for the evolution of genomic imprinting. (1) Imprinting may be advantageous in itself when imprinted and unimprinted alleles of a locus confer different phenotypes. If a segment of DNA is imprinted in the gametes of one sex but not in those of the other, it might lead to effects correlated with sexual dimorphism. More fundamentally, in certain organisms, sex determination might have evolved because of imprinting. When imprinting leads to chromosome elimination or inactivation and occurs in some embryos but not in others, two classes of embryos, differing in the number of functional gene copies, would result. A model for sex determination based on inequality in the actual or effective copy-number of particular noncoding, regulatory sequences of DNA has been proposed (Chandra, Proc. natn. Acad. Sci. U.S.A. 82. 1165–1169 and 6947–6949, 1985). Maternal control of offspring sex is another possible consequence of imprinting; this would indicate a potential role for imprinting in sex ratio evolution. (2) Genes responsible for imprinting may have pleiotropic effects and they may have been selected for reasons other than their imprinting ability. Lack of evidence precludes further consideration of this possibility. (3) Imprinting could have co-evolved with other traits. For instance, gamete-specific imprinting could lead to a lowered fitness of androgenetic or gynogenetic diploids relative to the fitness of ‘normal’ diploids. This in turn would reinforce the evolution of anisogamy. The reversibility of imprinting raises the possibility of occasional incomplete or improper erasure. If the site of imprinting is the egg – as appears to be the case with the human X (Chandra and Brown, Nature 253. 165–168, 1975) – either improper imprinting or improper erasure could lead to unusual patterns of inheritance (as in the fragile-X syndrome) or fitness effects skipping generations.