Synthetic hydrogels formed by thiol-ene crosslinking of vinyl sulfone-functional poly(methyl vinyl ether-alt-maleic acid) with α,ω-dithio-polyethyleneglycol.

Polymer hydrogels formed by rapid thiol-ene coupling of macromolecular gel formers can offer access to versatile new matrices. This paper describes the efficient synthesis of cysteamine vinyl sulfone (CVS) trifluoroacetate, and its incorporation into poly(methyl vinyl ether-alt-maleic anhydride) (PMMAn) to form a series of CVS-functionalized poly(methyl vinyl ether-alt-maleic acid) polymers (PMM-CVSx) containing 10 to 30 mol% pendant vinyl sulfone groups. Aqueous mixtures of these PMM-CVS and a dithiol crosslinker, α,ω-dithio-polyethyleneglycol (HS-PEG-SH, Mn = 1 kDa), gelled through crosslinking by Michael addition within seconds to minutes, depending on pH, degree of functionalization, and polymer loading. Gelation efficiency, Young's modulus, equilibrium swelling and hydrolytic stability are described, and step-wise hydrogel post-functionalization with a small molecule thiol, cysteamine, was demonstrated. Cytocompatibility of these crosslinked hydrogels towards entrapped 3T3 fibroblasts was confirmed using a live/dead fluorescence assay.

Inhibition of telomerase limits the growth of human cancer cells.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1.

Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal-regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.

Cell cycle arrest by Vpr in HIV-1 virions and insensitivity to antiretroviral agents.

Expression of human immunodeficiency virus-type 1 (HIV-1) Vpr after productive infection of T cells induces cell cycle arrest in the G2 phase of the cell cycle. In the absence of de novo expression, HIV-1 Vpr packaged into virions still induced cell cycle arrest. Naturally noninfectious virus or virus rendered defective for infection by reverse transcriptase or protease inhibitors were capable of inducing Vpr-mediated cell cycle arrest. These results suggest a model whereby both infectious and noninfectious virions in vivo, such as those surrounding follicular dendritic cells, participate in immune suppression.

Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells.

The UV-sensitive Chinese hamster ovary (CHO) cell line UV5, which is defective in the incision step of nucleotide excision repair, was used to identify and clone a complementing human gene, ERCC2, and to study the repair process. Genomic DNA from a human-hamster hybrid cell line was sheared and cotransferred with pSV2gpt plasmid DNA into UV5 cells to obtain five primary transformants. Transfer of sheared DNA from one primary transformant resulted in a secondary transformant expressing both gpt and ERCC2. The human repair gene was identified with a probe for Alu-family repetitive sequences. For most primary, secondary, and cosmid transformants, survival after UV exposure showed a return to wild-type levels of resistance. The levels of UV-induced mutation at the aprt locus for secondary and cosmid transformants varied from 50 to 130% of the wild-type level. Measurements of the initial rate of UV-induced strand incision by alkaline elution indicated that, whereas the UV5 rate was 3% of the wild-type level, rates of cosmid-transformed lines were similar to that of the wild type, and the secondary transformant rate was about 165% of the wild-type rate. Analysis of overlapping cosmids determined that ERCC2 is between 15.5 and 20 kilobases and identified a closely linked gpt gene. Cosmids were obtained with functional copies of both ERCC2 and gpt. ERCC2 corrects only the first of the five CHO complementation groups of incision-defective mutants.

Glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density.

Glucocorticoid administration to mice results in a rapid loss of bone mineral density due to an imbalance in osteoblast and osteoclast numbers. Whereas excess glucocorticoids reduce both osteoblast and osteoclast precursors, cancellous osteoclast number surprisingly does not decrease as does osteoblast number, presumably due to the ability of glucocorticoids to promote osteoclast life span. Whether glucocorticoids act directly on osteoclasts in vivo to promote their life span and whether this contributes to the rapid loss of bone with glucocorticoid excess remains unknown. To determine the direct effects of glucocorticoids on osteoclasts in vivo, we expressed 11beta-hydroxysteroid dehydrogenase type 2, an enzyme that inactivates glucocorticoids, specifically in the osteoclasts of transgenic mice using the tartrate-resistant acid phosphatase promoter. Bone mass, geometry, and histomorphometry were similar in untreated wild-type and transgenic animals. Glucocorticoid administration for 7 d caused equivalent increases in cancellous osteoblast apoptosis, and equivalent decreases in osteoblasts, osteoid, and bone formation, in wild-type and transgenic mice. In contrast, glucocorticoids stimulated expression of the mRNA for calcitonin receptor, an osteoclast product, in wild-type but not transgenic mice. Consistent with the previous finding that glucocorticoids decrease osteoclast precursors and prolong osteoclast life span, glucocorticoids decreased cancellous osteoclast number in the transgenic mice but not wild-type mice. In accord with this decrease in osteoclast number, the loss of bone density observed in wild-type mice was strikingly prevented in transgenic mice. These results demonstrate for the first time that the early, rapid loss of bone caused by glucocorticoid excess results from direct actions on osteoclasts.

Infrared Spectroscopic Imaging of the Biochemical Modifications Induced in the Cerebellum of the Niemann-Pick type C Mouse.

We have applied Fourier transform infrared (IR) spectroscopic imaging to the investigation of the neuropathologic effects of a genetic lipid storage disease, Niemann-Pick type C (NPC). Tissue sections both from the cerebella of a strain of BALB/c mice that demonstrated morphology and pathology of the human disease and from control animals were used. These samples were analyzed by standard histopathological procedures as well as this new IR imaging approach. The IR absorbance images exhibit contrast based on biochemical variations and allow for the identification of the cellular layers within the tissue samples. Furthermore, these images provide a qualitative description of the localized biochemical differences existing between the diseased and control tissue in the absence of histological staining. Statistical analyses of the IR spectra extracted from individual cell layers of the imaging data sets provide concise quantitative descriptions of these biochemical changes. The results indicate that lipid is depleted specifically in the white matter of the NPC mouse in comparison to the control samples. Minor differences were noted for the granular layers, but no significant differences were observed in the molecular layers of the cerebellar tissue. These changes are consistent with significant demyelination within the cerebellum of the NPC mouse. © 1999 Society of Photo-Optical Instrumentation Engineers.

Sexual dimorphism and ontogenetic allometry of soft tissues in Rattus norvegicus.

Most studies of sexual dimorphism in mammals focus on overall body size. However, relatively little is known about the differences in growth trajectories that produce dimorphism in organ and muscle size. We weighed six organs and four muscles in Rattus norvegicus to determine what heterochronic and allometric scaling differences exist between the sexes. This cross-sectional growth study included 113 males and 109 females with ages ranging from birth to 200 days of age. All muscle and organ weights were ultimately greater in males than in females, because males grew for a longer period of time, had a greater maximum rate of growth, and spent more time near the maximum rate. No ontogenetic scaling differences existed between the sexes in organ weight except for lungs and gonads. During growth, organ weights were negatively allometric to body weight. No scaling differences relative to body weight existed between the sexes for muscles; however, there was variation in the allometric relations among muscles relative to body weight. Sexual dimorphism in muscles and organs appears to be a size difference resulting from differences in the duration and rates of growth.

Two new species of Acanthobothrium in Narcine entemedor (Rajiformes: Narcinidae) from the northwest coast of Guanacaste Peninsula, Costa Rica.

This paper describes 2 new species of Acanthobothrium collected in Narcine entemedor from Cuajiniquil, Guanacaste Province, Costa Rica (10 degrees 57'N, 85 degrees 42'W). Acanthobothrium franus n. sp. averages 27 mm long, composed of 110 proglottides, has bothridial hooks 344-469 microns long, and 24-56 testes per proglottis. This new species resembles Acanthobothrium colombianum, Acanthobothrium coquimbensis. Acanthobothrium dujardini, Acanthobothrium lineatum, Acanthobothrium lintoni, and Acanthobothrium paulum. The new species differs from these 6 species by having a relatively shorter cirrus sac length not reaching the middle region of the proglottis. Additionally, A. franus differs from these species by having longer bothridia (627-1,408 microns vs. 299-391 microns for A. colombianum, 312-480 microns for A. coquimbensis, 240-560 microns for A. dujardini, 275-624 microns for A. lineatum, 389-720 microns for A. lintoni, and 300-880 microns for A. paulum), and larger bothridial hooks (344-469 microns vs. 175-193 microns, 120-192 microns, 180-210 microns, 118-216 microns, 108-230 microns, and 104-229 microns, respectively). Acanthobothrium inbiorium n. sp. averages 59 mm long, composed of 198 proglottides, has bothridial hooks 95-120 microns long, and possesses 44-73 testes per proglottis. Among species of Acanthobothrium, the new species resembles Acanthobothrium electricolum, Acanthobothrium dasybati, Acanthobothrium dighaensis, Acanthobothrium icelandicum, Acanthobothrium indicum, Acanthobothrium microcephalum, and Acanthobothrium wedli. The new species closely, resembles A. dasybati, but differs from that species in average strobila length and number of proglottides (58 microns long and 198 proglottides in A. inbiorium vs. 20 and 80 in A. dasybati, respectively). The new species can be distinguished from A. electricolum by having a wider scolex (450-900 microns vs. 189-252 microns), from A. dighaensis by having a narrower scolex (450-900 vs. 1,050-1,429), and from A. indicum by average strobilar length and number of proglottides (58 mm and 198 for A. inbiorium vs. 25 mm and 145 for A. indicum). Finally, A. inbiorium differs from A. icelandicum by having a shorter cirrus sac (122-285 for A. inbiorium vs. 380-410 for A. icelandicum), and A. microcephalum and A. wedli by having longer bothridia (an average of 603 microns vs. 447 microns for A. microcephalum and 350 microns for A. wedli), and fewer testes per proglottis (44-73 vs. 105-115 and 80-100, respectively). Morphological similarities suggest that some components of the eastern Pacific fauna of Acanthobothrium might share historical associations with the Caribbean and the western Pacific/Indian Ocean fauna.

Meeting a person with AIDS in the classroom: an evaluation.

Meeting a person with AIDS in the classroom was evaluated to determine if it had an impact on students' perceived susceptibility to HIV infection and attitudes toward persons with AIDS. The meeting was incorporated into the Grade 9 AIDS education program of a school district in Nova Scotia. Four schools participated in this study. Two schools were randomly assigned to the treatment group, which met the person with AIDS, and the remaining two schools formed the comparison group. Measures of the two attitudinal variables were collected using a self-report questionnaire that was administered both prior to and two weeks after the educational intervention. Meeting a person with AIDS in the classroom had no measurable impact on students perceived susceptibility to HIV infection nor on their attitudes toward persons with AIDS. Suggestions for using the educational intervention more effectively and for further research are made.

The surgical overcorrection of intermittent exotropia.

Many strabismus surgeons recommend an initial surgical overcorrection for intermittent exotropia. Others caution against overcorrection because of possible nasal suppression and amblyopia in children, or because of possible diplopia in adults. We reviewed the records of 69 patients who were initially overcorrected following surgery for an intermittent exotropia. The mean postoperative follow-up was 3.1 years. Eight patients (11.6%) had a persistent overcorrection of 3 prism diopters or more and three patients (4.3%) had persistent diplopia. Patients with a persistent overcorrection had a greater mean age (P less than .02) and a greater mean initial overcorrection (P less than .005) compared with the patients who were not overcorrected 3 delta or more. No child lost stereoacuity or developed amblyopia due to the overcorrection.

Prism adaptation for esotropia with a distance-near disparity.

A series of 64 patients who had surgery for esotropia with a distance-near disparity of at least 10 prism diopters was reviewed. Thirty-three patients were prism adapted for their distance deviation (PA distance). Thirty-one patients were prism adapted for near deviation (PA near). Both groups were divided into responders and nonresponders. In the PA distance group, 22 (67%) patients were responders. All responders had surgery for their prism-adapted angle. Postoperatively, 19 (86%) responders had fusion. Thirteen (68%) required bifocals to maintain fusion. In the PA near group, 21 (68%) patients were responders. All responders had surgery for their prism-adapted angle. Ninety-four percent of responders had fusion postoperatively. None needed bifocals for fusion postoperatively and none were overcorrected. The results show that those patients who were prism adapted for their near angle, responded with fusion, and had surgery for their full amount of esotropia at prism response obtained better postoperative fusion, without the need for a bifocal at near and without overcorrection at distance.

The diagnosis of amblyopia in cross-fixation.

Customarily, it is taught that cross-fixation, in a patient with congenital esotropia, obviates the development of amblyopia. However, our clinical experience has shown significant amblyopia in 50% of cross-fixators. In our hands, the diagnosis of amblyopia is made based on the point at which alternation of fixation takes place. By this method, if there is equal visual acuity, alternation will occur at the midline with each eye. If amblyopia exists, the sound eye will continue to follow the target beyond midline, into abduction, before the poorer seeing eye picks up fixation. In order to test the reliability of this method, using Teller acuity cards as the standard, we compared estimates of objective and subjective vision in 25 consecutive patients with congenital esotropia and cross-fixation. Our findings suggest that there may be a significant prevalence of amblyopia in cross- fixating patients and that the point at which alternation of fixation occurs is a reliable means of detecting a difference in visual acuity between the two eyes.

FEN1 contributes to telomere stability in ALT-positive tumor cells.

Abrogation of telomere stability through loss-of-function mutations in telomere binding proteins contributes to genomic instability and cancer progression. Recently, Flap endonuclease 1 (FEN1) was shown to contribute to telomere stability in human cells that had not yet activated a telomere maintenance mechanism, suggesting that abrogation of FEN1 function influences the transformation process by compromising telomere stability and driving genomic instability. Here, we analyse the telomeres in human cancer cells following FEN1 depletion. We show that FEN1 is required for telomere stability in cells that rely on the alternative lengthening of telomere (ALT) mechanism. Indeed, FEN1 depletion resulted in telomere dysfunction, characterized by formation of telomere dysfunction-induced foci (TIFs) and end-to-end fusions in ALT-positive cells. In contrast, no telomere phenotype was observed in telomerase-positive cells on FEN1 depletion, suggesting that ongoing telomerase activity protected telomeres. In consonance with this, we found that expression of the catalytic component of telomerase (hTERT) but not an inactive allele rescued telomere dysfunction on FEN1 depletion in ALT cells. Our data suggest that mutations that arise in FEN1 affect telomere stability and genome fidelity by promoting telomere fusions and anaphase-bridge-breakage cycles, which further drive genome instability and thereby contribute to the transformation process.

Telomerase and human tumorigenesis.

Human cancer cells, unlike their normal counterparts, have shed the molecular restraints to limited cell growth and are immortal. Exactly how cancer cells manage this at the molecular level is beginning to be understood. Human cells must overcome two barriers to cellular proliferation. The first barrier, referred to as senescence, minimally involves the p53 and Rb tumor-suppressor pathways. Inactivation of these pathways results in some extension of lifespan. However, inactivation of these pathways is insufficient for immortalization. As normal cells undergo repeated rounds of DNA replication, their telomeres shorten due to the inability of traditional DNA polymerases to completely replicate the end of the chromosomal DNA. This shortening continues until the cells reach a second proliferative block referred to as crisis, which is characterized by chromosomal instability, end-to-end fusions, and cell death. Stabilization of the telomeric DNA through either telomerase activation or the activation of the alternative mechanism of telomere maintenance (ALT) is essential if the cells are to survive and proliferate indefinitely. Conversely, loss of telomere stabilization by an already-immortalized cell results in loss of immortality and cell death. Together this indicates that telomere maintenance is a critical component of immortality. In this review we attempt to describe our current understanding of the role of telomere maintenance in senescence, crisis, and tumorigenesis.

Biochemical and genetic analysis of the Chinese hamster mutants irs1 and irs2 and their comparison to cultured ataxia telangiectasia cells.

Two mutants of the Chinese hamster cell line V79-4 (irs1 and irs2) were previously isolated on the basis of their hypersensitivity (2- to 3-fold) to cell inactivation by ionizing radiation. One of these mutants, irs1, displays an unusual phenotype of cross-sensitivity to other varied genotoxic agents including UV light (2- to 3-fold), ethyl methanesulphonate (approximately 10-fold) and mitomycin C (approximately 60-fold). The possibility that these sensitivities might be due to more than one gene mutation in irs1 was investigated. Hybrids formed between irs1 and human lymphocytes were isolated in which the mitomycin C (MMC) sensitivity of irs1 was corrected by complementing human chromosomal material. These MMC-resistant hybrids and their subclones also showed concordant correction of the gamma-ray, UV and EMS sensitivities of irs1, suggesting that a single gene defect is most likely responsible for the phenotype of irs1. In addition it was shown that the MMC-sensitivity of irs1 is complemented by four CHO cell mutants (UV20, UV41, UV-1 and irs1SF), which also display extreme sensitivity to MMC. Mutants irs1, irs1SF and UV-1 define three new complementation groups for MMC sensitivity. The biochemical nature of the ionizing radiation sensitivity of irs1 and irs2 was also investigated. The production and repair of DNA single- and double-strand breaks were studied using the techniques of alkaline and neutral elution, respectively. Irs1 and irs2 both showed repair kinetics for each lesion that are indistinguishable from wild-type. Analysis of the rate of DNA synthesis following gamma-irradiation showed irs1 to have a dose-dependent inhibition similar to that of wild-type.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuromyotonia in hereditary motor neuropathy.

Two siblings with a distal motor neuropathy experienced cramping and difficulty in relaxing their muscles after voluntary contraction. Electromyographic recordings at rest revealed repetitive high voltage spontaneous electrical discharges that were accentuated after voluntary contraction and during ischaemia. Regional neuromuscular blockage with curare indicated hyperexcitability of peripheral nerve fibres and nerve block suggested that the ectopic activity originated in proximal segments of the nerve. Symptoms were improved with diphenylhydantoin, carbamazepine and tocainide.

Lentiviral delivery of HIV-1 Vpr protein induces apoptosis in transformed cells.

Most current anticancer therapies act by inducing tumor cell stasis followed by apoptosis. HIV-1 Vpr effectively induces apoptosis of T cells after arrest of cells at a G(2)/M checkpoint. Here, we investigated whether this property of Vpr could be exploited for use as a potential anticancer agent. As a potentially safer alternative to transfer of genes encoding Vpr, we developed a method to efficiently introduce Vpr protein directly into cells. Vpr packaged into HIV-1 virions lacking a genome induced efficient cell cycle arrest and apoptosis. Introduction of Vpr into tumor cell lines of various tissue origin, including those bearing predisposing mutations in p53, XPA, and hMLH1, induced cell cycle arrest and apoptosis with high efficiency. Significantly, apoptosis mediated by virion-associated Vpr was more effective on rapidly dividing cells compared with slow-growing cells, thus, in concept, providing a potential differential effect between some types of tumor cells and surrounding normal cells. This model system provides a rationale and proof of concept for the development of potential cancer therapeutic agents based on the growth-arresting and apoptotic properties of Vpr.

Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest.

The human immunodeficiency virus type 1 (HIV-1) vpr gene encodes a protein which induces arrest of cells in the G2 phase of the cell cycle. Here, we demonstrate that following the arrest of cells in G2, Vpr induces apoptosis in human fibroblasts, T cells, and primary peripheral blood lymphocytes. Analysis of various mutations in the vpr gene revealed that the extent of Vpr-induced G2 arrest correlated with the levels of apoptosis. However, the alleviation of Vpr-induced G2 arrest by treatment with the drug pentoxifylline did not abrogate apoptosis. Together these studies indicate that induction of G2 arrest, but not necessarily continued arrest in G2, was required for Vpr-induced apoptosis to occur. Finally, Vpr-induced G2 arrest has previously been correlated with inactivation of the Cdc2 kinase. Some models of apoptosis have demonstrated a requirement for active Cdc2 kinase for apoptosis to occur. Here we show that accumulation of the hypophosphorylated or active form of the Cdc2 kinase is not required for Vpr-induced apoptosis. These studies indicate that Vpr is capable of inducing apoptosis, and we propose that both the initial arrest of cells and subsequent apoptosis may contribute to CD4 cell depletion in HIV-1 disease.