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The actin cytoskeleton assembles into branched networks or bundles to generate mechanical force for critical cellular processes such as establishment of polarity, adhesion, and migration. Stress fibers (SFs) are contractile actomyosin structures that physically couple to the extracellular matrix through integrin-based focal adhesions (FAs), thereby transmitting force into and across the cell. Recently, LIN-11, Isl1, and MEC-3 (LIM) domain proteins have been implicated in mediating this cytoskeletal mechanotransduction. Among the more well-studied LIM domain adapter proteins is zyxin, a dynamic component of both FAs and SFs. Here we discuss recent research detailing the mechanisms by which SFs adjust their structure and composition to balance mechanical forces and suggest ways that zyxin and other LIM domain proteins mediate mechanoresponse., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

One striking feature of spontaneous autoimmune diabetes is the prototypic formation of lymphoid follicular structures within the pancreas. Lymphotoxin (LT) has been shown to play an important role in the formation of lymphoid follicles in the spleen. To explore the potential role of LT-mediated microenvironment in the pathogenesis of insulin-dependent diabetes mellitus (IDDM), an LTbeta receptor-immunoglobulin fusion protein (LTbetaR-Ig) was administered to nonobese diabetic mice. Early treatment with LTbetaR-Ig prevented insulitis and IDDM, suggesting that LT plays a critical role in the insulitis development. LTbetaR-Ig treatment at a late stage of the disease also dramatically reversed insulitis and prevented diabetes. Moreover, LTbetaR-Ig treatment prevented the development of IDDM by diabetogenic T cells in an adoptive transfer model. Thus, LTbetaR-Ig can disassemble the well established lymphoid microenvironment in the islets, which is required for the development and progression of IDDM.

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

Sox9 plays a crucial role in chondrogenesis. It encodes an HMG-domain transcription factor that activates an enhancer in the gene for type II collagen (Col2a1), a principal cartilage matrix protein. We have characterized the temporal pattern of Sox9 RNA expression in micromass culture, a widely used in vitro model for the analysis of embryonic cartilage differentiation. Cultures were prepared from distal subridge mesenchyme of the stage 24/25 chick embryo wing bud, which undergoes uniform chondrogenic differentiation in vitro. The early "prechondrogenic" phase of culture was characterized by the activation of Sox9 RNA expression, which preceded detectable upregulation of Col2a1 transcription. Sox9 RNA levels peaked between 20 and 65 h of culture, a phase of progressive Col2a1 transcript accumulation, then declined in the mature cartilage of 120-h cultures. Staurosporine treatment enhanced chondrogenesis in micromass culture by inducing a rapid quantitative increase in Sox9 transcript levels. However, PMA, a phorbol ester that inhibits Col2a1 expression and chondrocyte differentiation, had an unexpectedly modest effect on Sox9 RNA accumulation., (Copyright 2000 Academic Press.)

Preformed transposase-transposon complexes called 'Transposomes' have been electroporated into bacterial cells. The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo. The transposition efficiency of a Transposome containing a kanamycin marker was between 1.0 x 10(4) and 1.0 x 10(7) kanamycin resistant clones per microgram of transposon DNA in three gram-negative enteric bacterial species. Transposon integration sites were examined by direct genome sequencing of chromosomal DNA. Genomic DNA was isolated from transposition clones and directly cycle sequenced with primers specific for the ends of the transposon. The precise location of genome interruption for a transposition clone was identified by homology to known genes or sequences. Mutant phenotypes were rapidly correlated with genomic insertions sites.

Theiler's murine encephalomyelitis virus is an endemic murine pathogen that induces a demyelinating disease of the central nervous system in susceptible mouse strains. The disease is characterized by central nervous system mononuclear cell infiltration and presents as chronic, progressive paralysis. The expression of CC and C-x-C chemokines in the central nervous system of Theiler's murine encephalomyelitis virus-infected mice was examined throughout the disease course by ELISA and RT - PCR analysis. Central nervous system expression of MCP-1 and MIP-1alpha protein was evident by day 11 post Theiler's murine encephalomyelitis virus infection of SJL mice and continued throughout disease progression. MIP-1alpha, RANTES, MCP-1, C10, IP-10, and MIP-1beta mRNA was specifically expressed in the central nervous system and not the periphery following Theiler's murine encephalomyelitis virus infection. This was associated with development of clinical disease. These data suggest that the expression of multiple chemokines at particular times following viral infection is associated with demyelinating disease.

Base excision sequence scanning (BESS) is a new polymerase chain reaction (PCR)-based mutation scanning method that locates and identifies all DNA mutations. The BESS method consists of two procedures that generate "T" (BESS T-Scan) and "G" ladders (BESS G-Tracker) analogous to T and G ladders of dideoxy sequencing. The BESS procedures are simple to perform and require no special equipment or gels, no reaction optimization beyond PCR, and no heteroduplex formation. The samples are analyzed on standard sequencing gels or on automated DNA sequencers, and the data produced are easy to interpret, requiring a simple comparison of the sequence of normal and mutant DNA. The BESS method is versatile, having applications not only for mutation detection, but also single nucleotide polymorphism (SNP) discovery and analysis, DNA fingerprinting (including viral and bacterial typing), and clone identification. In this study, we utilize BESS in two of these applications: detection of a point mutation in BRCA1, and DNA typing of human papilloma virus (HPV).

Ethanol is a well-recognized teratogen in vertebrates that can perturb the development of the facial primordia and various other embryonic structures. However,the mechanisms underlying alcohol's effects on embryogenesis are currently unclear. Recent evidence suggests that the cranial neural crest, which forms the entire facial skeleton, may be a particularly sensitive target of ethanol teratogenicity. In the present study we have examined the influence of in vitro ethanol exposure on cartilage differentiation in micromass cultures of mesenchymal cells isolated from the various facial primordia (maxillary, mandibular, frontonasal, and hyoid processes) of the stage 24 chick embryo. In all four populations of facial mesenchyme, exposure to 1-1.5% ethanol promoted marked increases in Alcian blue-positive cartilage matrix formation, a rise in 35SO4 accumulation into matrix glycosaminoglycans, and enhanced expression of cartilage-characteristic type II collagen and aggrecan gene transcripts. In frontonasal and mandibular mesenchyme cultures, which undergo extensive spontaneous cartilage formation, ethanol treatment quantitatively elevated both matrix production and cartilage-specific gene transcript expression. In cultures of maxillary process and hyoid arch mesenchyme, which form little or no cartilage spontaneously, ethanol exposure induced the formation of chondrogenic cell aggregates and the appearance of aggrecan and type II collagen mRNAs. These actions were not restricted to ethanol, since tertiary butanol treatment also enhanced cartilage differentiation in facial mesenchyme cultures. Our findings demonstrate a potent stimulatory effect of alcohol on the differentiation of prechondrogenic mesenchyme of the facial primordia. Further analysis of this phenomenon might yield insight into the developmental mechanisms underlying the facial dysmorphologies associated with embryonic ethanol exposure.

Purpose: To evaluate the role of cell-mediated immunity in the stability of ocular adenovirus-mediated transgene expression., Methods: Adenovirus (4 x 10(6) pfu) containing lacZ (Ad.CMVlacZ) was injected intravitreally or subretinally into one or both eyes of immunocompetent (+/+) and immunocompromised (nu/nu) CD-1 mice. Control eyes received injections of saline. Additional +/+ mice received subretinal injections of Ad.CMVlacZ with coadministration of 200 microg of human immunoglobulin (Ig) G or CTLA4Ig by intraperitoneal, intravitreal, or subretinal injection. The mice were killed at various times after injection, and their eyes were examined histologically and immunohistochemically., Results: LacZ expression was extended from 1 week to more than 5 weeks in the corneal endothelium, iris, and trabecular meshwork of nu/nu mice compared with time of expression in +/+ mice when adenovirus was administered intravitreally. In contrast, subretinal injection resulted in only a minimal increase in transgene stability in nu/nu mice compared with that in +/+ mice. Neither systemic nor intraocular administration of IgG or CTLA4Ig affected the stability of lacZ expression in the retina or retinal pigment epithelium after subretinal injection in +/+ mice. Immunoglobulin G and CTLA4Ig enhanced the stability of transgene expression in the trabecular meshwork., Conclusions: A T-cell-mediated immune response appears to play a role in the loss of adenovirus-mediated lacZ expression after intravitreal but not after subretinal injection. This result implies that the subretinal space is an immune-privileged site and a favorable site for gene transfer.

Studies of neural, hepatic, and other cells have demonstrated that in vitro ethanol exposure can influence a variety of membrane-associated signaling mechanisms. These include processes such as receptor-kinase phosphorylation, adenylate cyclase and protein kinase C activation, and prostaglandin production that have been implicated as critical regulators of chondrocyte differentiation during embryonic limb development. The potential for ethanol to affect signaling mechanisms controlling chondrogenesis in the developing limb, together with its known ability to promote congenital skeletal deformities in vivo, prompted us to examine whether chronic alcohol exposure could influence cartilage differentiation in cultures of prechondrogenic mesenchyme cells isolated from limb buds of stage 23-25 chick embryos. We have made the novel and surprising finding that ethanol is a potent stimulant of in vitro chondrogenesis at both pre- and posttranslational levels. In high-density cultures of embryonic limb mesenchyme cells, which spontaneously undergo extensive cartilage differentiation, the presence of ethanol in the culture medium promoted increased Alcian-blue-positive cartilage matrix production, a quantitative rise in 35SO4 incorporation into matrix glycosaminoglycans (GAG), and the precocious accumulation of mRNAs for cartilage-characteristic type II collagen and aggrecan (cartilage proteoglycan). Stimulation of matrix GAG accumulation was maximal at a concentration of 2% ethanol (v/v), although a significant increase was elicited by as little as 0.5% ethanol (approximately 85 mM). The alcohol appears to directly influence differentiation of the chondrogenic progenitor cells of the limb, since ethanol elevated cartilage formation even in cultures prepared from distal subridge mesenchyme of stage 24/25 chick embryo wing buds, which is free of myogenic precursor cells. When limb mesenchyme cells were cultured at low density, which suppresses spontaneous chondrogenesis, ethanol exposure induced the expression of high levels of type II collagen and aggrecan mRNAs and promoted abundant cartilage matrix formation. These stimulatory effects were not specific to ethanol, since methanol, propanol, and tertiary butanol treatments also enhanced cartilage differentiation in embryonic limb mesenchyme cultures. Further investigations of the stimulatory effects of ethanol on in vitro chondrogenesis may provide insights into the mechanisms regulating chondrocyte differentiation during embryogenesis and the molecular basis of alcohol's teratogenic effects on skeletal morphogenesis.

Milligram quantities of ribozymes (Rzs) can be synthesized in vitro in reaction volumes of 1 mL or less using AmpliScribe T7 RNA polymerase kits to transcribe either linear plasmids or oligodeoxynucleotide templates. Model hammerhead Rzs were synthesized that specifically cleave RNA-encoding chloramphenicol acetyl transferase sequences. Methods are presented for the transcription of Rzs of virtually any length and sequence composition at a fraction of the cost of chemical synthesis.

Cultures derived from a malignant glioma (U-87 MG) were treated with 3 mM dibutyryl cAMP. The treatment resulted in morphological differentiation of the cultures and a decrease in cell proliferation. Biochemically, dibutyryl cAMP treatment caused a general reduction in the concentration of neutral glycosphingolipids in the U-87 MG cells. The concentration of individual neutral glycosphingolipids in the untreated cells was 1.8- to 3.0-fold higher than in cells treated for 72 h with 3 mM dibutyryl cAMP. Cells were labeled with [3H]galactose to monitor synthesis of the neutral glycosphingolipids. Decreased synthesis was noted in cells treated with dibutyryl cAMP as compared with untreated cells as indicated by decreased uptake of [3H]galactose label. The ganglioside composition of the cells was essentially unchanged after dibutyryl cAMP treatment.

In previous work, we observed the presence of substantially elevated levels of GM2 after Simian Virus 40 (SV-40) transformation of human fetal brain cells. This elevated level of GM2 contrasted with the reports of many other investigators who had often observed decreased levels of GM2 and a simplification of ganglioside pattern in various non-neural rodent cell lines. In order to determine if the increase in GM2 in the transformed human brain cells would also be found in transformed rodent brain cells, we analyzed ganglioside changes after transformation in mouse brain cell lines and observed the increase in GM3 and low levels or lack of GM2 usually noted in rodent SV-40 transformed cell lines. In addition, we analyzed changes after SV-40 transformation in three human fibroblast lines and found that all three lines contained substantially elevated levels of GM2 after SV-40 transformation. As a result of this study, our earlier work on SV-40 transformed human brain cells, and occasional other reports of high levels of GM2 in human SV-40 transformed cell lines, elevated levels of GM2 may be considered a marker for SV-40 transformed human cells of both fibroblastic and neural origin.

Soluble proteins that reside in the lumen of the endoplasmic reticulum are known to have at their carboxyterminus the tetrapeptides KDEL or HDEL. In yeast and mammalian cells, these tetrapeptides function as endoplasmic reticulum (ER)-retention signals. To determine the effect of an artificially-introduced KDEL sequence at the exact carboxyterminus of a plant secretory protein, we modified the gene of the vacuolar protein phytohemagglutinin-L (PHA) so that the amino-acid sequence would end in LNKDEL rather than LNKIL, and expressed the modified gene in transgenic tobacco with a seed-specific promoter. Analysis of the glycans of PHA showed that most of the control PHA had one endoglycosidase H-sensitive and one endoglycosidase H-resistant glycan, indicating that it had been processed in the Golgi complex. On the other hand, a substantial portion of the PHA-KDEL (about 75% at mid-maturation and 50% in mature seeds) had two endoglycosidase H-sensitive glycans. Phytohemagglutinin with two endoglycosidase H-sensitive glycans is normally found in the ER. Using immunocytochemistry we found that a substantial portion of the PHA-KDEL was present in the ER or accumulated in the nuclear envelope while the remainder was found in the protein storage vacuoles (protein bodies). We interpret these data to indicate that carboxyterminal KDEL functions as an ER retention-retardation signal and causes protein to accumulate in the nuclear envelope as well as in the ER. The incomplete ER retention of this protein which is modified at the exact carboxyterminus may indicate that structural features other than carboxyterminal KDEL are important if complete ER retention is to be achieved.Mention of trademark, proprietary product, or vendor, does not constitute a guarantee or warrenty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

A novel enzyme for DNA dephosphorylation, HK phosphatase, is completely and irreversibly inactivated at 65 degrees C. HK phosphatase treatment of BamHI-digested pBR322 reduces nonrecombinants in cloning experiments to approx. 5% of transformant colonies.

There have been two trends within the physician housestaff movement: increased acceptance of collective bargaining and unions, and a shift from narrower economic to broader political demands, including some involving patient care. Case studies of politically active housestaff associations in New York, Chicago, and Los Angeles are used to examine the emergence of "patient-care" demands and their compatibility with collective bargaining frameworks. As house-staff have become principal providers of care to indigent populations in public hospitals, and economic cutbacks have endangered service as well as the positions of physicians, patient-care demands arise and become infused with demands for participation and control in decision-making. Common factors in the politicization of housestaff have been the contribution of activists of the sixties as leaders, and the impact of fiscal crisis and economic retrenchment in the seventies. However, the emergence and resolution of these issues has differed depending upon legal, political, historical, and organizational variations. In general, patient-care issues are supported by housestaff when they dovetail with housestaff interests. However, physician interests can diverge from those of patients, as in the case of manpower redistribution. On the whole, wages and benefits have done better than educational or patient-care demands. Educational demands have met with counterattack, and patient care, limited by the traditional scope of collective bargaining, has had to evolve indirectly, and has been hurt by long-term economic trends. Finally, national housestaff organization is limited by the wide-ranging politics and ideas of diverse regional organizations which represent different types of training institutions and career orientations.

In vitro mutagenesis was used to supplement the sulfur amino acid codon content of a gene encoding β-phaseolin, a Phaseolus vulgaris storage protein. The number of methionine codons in the phaseolin gene was increased from three to nine by insertion of a 45 base pair (bp) synthetic duplex. Either modified or normal phaseolin genes were integrated into the genome of tobacco plants through Agrobacterium tumefaciens-mediated transformation. Although similar levels of phaseolin RNA are detected in seeds of plants transformed with either the normal or modified (himet) gene, the quantity of himet protein is consistently much lower than normal β-phaseolin. Himet phaseolin is expressed in a temporal- and organ-specific fashion, and is N-glycosylated and assembled into trimers in the manner of normal phaseolin. After germination, both types of phaseolin are hydrolyzed, but the himet protein is more quickly degraded. Electron microscopic immunocytochemical observations of developing seeds indicate that the himet protein is primarily localized in the endoplasmic reticulum (ER) and in Golgi apparatus secretion vesicles. Himet phaseolin is absent from protein storage vacuoles, termed protein bodies, where normal phaseolin is deposited in transgenic tobacco. We interpret the immunocytochemical data to indicate that himet phasolin is transported through the ER and Golgi apparatus and is then degraded in Golgi secretion vesicles or the protein bodies.

Cultured cells derived from Tay-Sachs disease (TSD) fetal cerebellum were shown to accumulate GM2 ganglioside when compared with control cultures. In contrast, fibroblasts derived from TSD fetal lung do not contain GM2. GM2 was identified by thin-layer chromatography (TLC) and confirmed by gas-liquid chromatography (GLC). Unlike fetal TSD brain, the cell cultures established from fetal TSD brain have high concentrations of globoside and GD3 and little or no asialo GM2(GA2). The GM2 and related glycosphingolipids in these cultured cells contain a high percentage of C24:0 and C24:1 fatty acids. In spite of these differences, this TSD cell strain is unique in that it accumulates GM2, and can therefore serve as a useful in vitro model for the study of TSD.

Human cells transformed by Simian virus 40 (SV40) usually show little or no tumor formation after implantation in nude mice. In long-term studies, however, we have observed that 127 to 366 days after subcutaneous injection of SV40-transformed human meningioma cells (KJ-M2-T) into nude mice (Nu/Cox), 6 of 15 animals developed lymphomas or fibrosarcomas, usually at the site of inoculation. The induced tumors were of murine origin and were positive for SV40-T antigen. Chromosome analysis and G11 staining revealed no evidence of hybridization between human and mouse cells. No spontaneous shedding of SV40 was noted with KJ-M2-T cells in vitro; however, SV40 could be rescued after fusion of KJ-M2-T with BS-C-1 monkey kidney cells, but not with L929 mouse fibroblasts. A parallel study using SV40-transformed human fetal brain cells failed to induce tumors in nude mice despite the demonstration that infectious SV40 could also be rescued from this line after fusion with BS-C-1 but not with L-929. Subcutaneous injection of 5 X 10(3) TCID's of SV40 (strain J436) into nude mice resulted in the induction of fibrosarcomas at the injection site in 6 of 15 mice after 273 to 396 days. The induction of malignant lymphomas after implantation of SV40 transformed cells contrasted with the development of fibrosarcomas after injection of free virus. This study suggests that after subcutaneous implantation into nude mice, some SV40-transformed human tumor cell lines can serve as vectors for transmitting SV40 to murine cells causing transformation and tumor development in the host animal.

Enzyme-replacement treatment for metabolic storage disorders has been widely studied using model cell culture systems. This study determines the long-term fate of human hexosaminidase A supplied to Tay-Sachs disease brain and lung cells. Hex A retention studies showed that the incorporated Hex A is retained in undiminished quantity by TSD lung cells maintained in stationary culture for 14 days. Tay-Sachs disease brain cells similarly followed for 28 days in stationary culture showed an initial reduction in Hex A for 3 days, after which the Hex A level stabilized and remained relatively constant for the next 25 days. Hexosaminidase B isoenzyme was found to accumulate in both cell lines during extended cultivation, despite the observation that significant amounts were excreted into the extracellular environment. The demonstration of long-term intracellular retention of exogenously supplied therapeutic enzyme by the target cells offers additional evidence for the feasibility of an enzyme-replacement approach for study and treatment of lysosomal storage disorders.

Lysolecithin and sphingomyelin may be readily separated from other phospholipids on thin-layer plates impregnated with silver nitrate using the solvent system chloroform-methanol-concentrated ammonium hydroxide 65:35:8. Quantitation is carried out by determining the phosphorus content of the developed bands. The minimum amount of phospholipid that can be quantitatively determined is that which contains 0.7 micro g of phosphorus. This method has been used for the determination of lysolecithin and sphingomyelin in the tissues of mice having foam-cell reticulosis.

The nucleotide sequences of eight partial and five full-length phaseolin cDNA clones show that phaseolin polypeptides are encoded by two distinct gene families which differ in their coding regions by the presence or absence of two different size direct repeats. The alpha-type phaseolin polypeptides are encoded by genes containing direct repeats which encode 14 additional amino acids. Aside from these differences, the alpha-and beta-type phaseolin genes show a high degree of homology (98%) which is consistent with these genes being derived from a common ancestral gene. Much of the heterogeneity found in the phaseolin polypeptides appears to be due to post-translational processing. Nucleotide sequence analysis demonstrates that the alpha-type genes contain only a few amino acid replacement substitutions and that the beta-type genes appear to contain no amino acid replacement substitutions. S1 nuclease mapping shows a complex pattern for transcriptional initiation of phaseolin mRNA. Hydropathy analysis shows that phaseolin polypeptides are predominately hydrophilic, and that the two N-glycosyl recognition sites are located in different hydropathic environments.

An RNA expression plasmid containing a maize zein storage protein gene was modified by the addition of an oligodeoxynucleotide duplex containing oligo(A30) in the RNA sense strand to the 3' flank of the gene. By linearizing the plasmid at either side of the oligo(A) region, transcripts with or without a poly(A) tail may be synthesized. Poly(A) addition dramatically increases the translation of SP6-transcribed zein transcripts in Xenopus laevis oocytes. We describe a derivative of pSP64, pSP64A, containing the oligo(A) region, which may be useful for the transcription of polyadenylated RNAs from any template DNA.