Molting in land crabs: stimulation by leg removal.

If the Bermuda land crab, Gecarcinus lateralis, loses numerous walking legs or both chelipeds, it undergoes almost immediate preparations for molting with attendant limb regeneration. Injections of the arthropod-molting hormone, ecdysterone, have no effect in either intact animals or those missing legs.

Malignant transformation and tumor promoter treatment increase levels of a transcript for a secreted glycoprotein.

The major excreted protein of transformed mouse fibroblasts, a secreted, mannose 6-phosphate-containing glycoprotein, is induced in nontransformed cells by a variety of transforming agents, by phorbol esters, and by platelet-derived growth factor. We report here the molecular cloning of the cDNA encoding this protein and demonstrate that its induction is a consequence of enhanced mRNA levels for major excreted protein in both tetradecanoyl phorbol acetate-treated 3T3 cells and 3T3 cells transformed by a variety of retroviruses or retroviral oncogenes. These results indicate that tumor promoters and retroviral transformation might share a common pathway of action in cultured cells and that major excreted protein is a molecular marker for the growth response of cells to these agents.

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

Expression of pregnancy-specific genes in preneoplastic mouse mammary tissues from virgin mice.

Experimentally induced breast cancer is often preceded by the appearance of preneoplastic lesions which possess the attributes of hyperplastic normal tissue. These lesions can be isolated and carried as stably transplantable outgrowth lines which continue to morphologically resemble differentiating mammary tissue (Medina, D. Methods Cancer Res., 7: 3-53, 1973). We established seven serially transplantable hyperplastic alveolar nodule (HAN) outgrowth lines from virgin mouse mammary tissues following induction by mouse mammary tumor virus, dimethylbenz(alpha)-anthracene, and/or pituitary isografts. The expression of mammary differentiation-specific casein genes was measured in these hyperplastic outgrowths by immunocytochemistry, specific radioimmune precipitation, and blot hybridization of total RNA. All seven HAN outgrowth lines were immunologically positive for casein both in situ and upon explant culture. Unlike explants from normal virgin mouse mammary gland, exposure to insulin, hydrocortisone, and prolactin induced an increase in casein synthesis in HAN explant cultures which was independent of DNA synthesis. [35S]Methionine-labeled polypeptides synthesized in explant cultures of HAN outgrowths freshly isolated from virgin hosts were analyzed by radioimmune precipitation and gel electrophoresis. This analysis demonstrated that all major species of casein, alpha (Mr 46,000), beta (Mr 27,000), and gamma (Mr 25,000), were constitutively (i.e., in the absence of lactogenic stimuli) expressed in these preneoplastic alveolar mammary outgrowths. In support of this observation, RNA homologous to beta- and alpha-casein cDNA probes was often detectable in total RNA preparations from freshly isolated fragments of HAN outgrowths. A second mammary differentiation specific gene product, alpha-lactalbumin, was also detected in HAN outgrowths both in situ and following explant culture. Enzymatically active alpha-lactalbumin was present in extracts of freshly isolated HAN outgrowth tissues and was detectable in these same tissues by immunoperoxidase. In general, alpha-lactalbumin synthesis was increased during explant culture in the presence of lactogenic hormones; however, in contrast to casein synthesis, insulin-hydrocortisone-prolactin-induced increase in alpha-lactalbumin production in vitro was occasionally dependent upon DNA synthesis as it is in explants from normal virgin mouse mammary tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rlGFBP-1, 2, or 3.

The bioavailability and action of the insulin-like growth factors (IGFs) are determined by specific IGF-binding proteins (IGFBP) to which they are complexed. Complementary DNA clones have been isolated that encode three related IGFBPs: human IGFBP-1 (hIGFBP-1), human IGFBP-3 (hIGFBP-3), and rat IGFBP-2 (rIGFBP-2). IGFBP-1 and IGFBP-3 are regulated differently in human plasma, suggesting that they have different functions. In order to study the molecular basis of the regulation of the different IGFBPs, we have identified a panel of rat cell lines that express a single predominant binding protein and developed an assay strategy to distinguish the different binding proteins. Proteins in conditioned medium were examined by ligand blotting, and by immunoprecipitation and immunoblotting using antibodies to rIGFBP-2 and hIGFBP-1; RNAs were hybridized to cDNA probes for rIGFBP-2 and hIGFBP-1. 1) C6 glial cells and B104 neuroblastoma cells express an approximately 40 kilodalton (kDa) glycosylated binding protein that most likely represents rIGFBP-3, the binding subunit of the 150 kDa IGF: binding protein complex in adult rat serum. The C6 and B104 binding proteins do not react with antibodies to rIGFBP-2, and RNAs from C6 and B104 cells do not hybridize to cDNA probes for rIGFBP-2 or hIGFBP-1. 2) BRL-3A, Clone 9, and TRL 12-15 cell lines derived from normal rat liver express rIGFBP-2, a 30 kDa nonglycosylated IGF-binding protein that is recognized by antibodies to rIGFBP-2 but not by antibodies to hIGFBP-1. RNAs from these cells hybridize to a rIGFBP-2 cDNA probe, but not to a hIGFBP-1 probe. 3) H35 rat hepatoma cells express a 30 kDa nonglycosylated IGFBP that is presumptively identified as rIGFBP-1. It does not react with antibodies to rIGFBP-2, but is recognized by polyclonal and monoclonal antibodies to hIGFBP-1. RNA from H35 cells hybridizes to a hIGFBP-1 cDNA probe, but not to a rIGFBP-2 probe. Expression of rIGFBP-1 by the H35 cell line has enabled us to establish and validate specific assays for this protein that allow us to study its regulation in intact rats. Identification of a panel of rat cell lines expressing specific IGFBPs should be useful in elucidating the molecular mechanisms of IGFBP regulation.

Rat C6 glial cells synthesize insulin-like growth factor I (IGF-I) and express IGF-I receptors and IGF-II/mannose 6-phosphate receptors.

We have used the rat C6 glial cell line as a model system to study the role of insulin-like growth factors (IGF) in neuroglial cells of the central nervous system (CNS). Northern blot analysis of C6 RNA demonstrated the presence of IGF-I mRNA and undetectable IGF-II mRNA. IGF-I and IGF-binding protein(s), but not IGF-II, were detected in C6 glial cell-conditioned medium. The level of IGF-I was 1-4 ng/ml in conditioned medium based on a human IGF-I standard. The immunoreactive IGF-I inhibited [125I]IGF-I binding to the IGF-I receptor on chick embryo fibroblasts and stimulated [3H]thymidine incorporation into chick embryo fibroblast DNA. Competitive binding and affinity cross-linking experiments using [125]IGF-I and [125I]IGF-II demonstrated the presence of IGF-I receptors (type I) and IGF-II/mannose 6-phosphate receptors (type II) on C6 glial cell membranes. An immunoglobulin (no. 3637) directed against the rat IGF-II receptor blocked the degradation of [125I]IGF-II added to C6 glial cells, presumably by blocking receptor-mediated internalization. We were unable to demonstrate an autocrine role for IGF in the C6 glial cell line, since [3H]thymidine incorporation into DNA was stimulated equally well by IGF-I-deficient rat serum and normal serum, and added IGF did not stimulate [3H]thymidine incorporation into DNA when tested alone or when added to IGF-I-deficient serum. We propose that neuroglial cell-derived IGF-I may serve as a paracrine growth stimulus in the central nervous system.

'Northern Cross' hybridization for rapid identification of exon-containing restriction fragments.

The Northern Cross method allows direct comparison of restriction digests of cDNA and genomic clones to RNA populations by a specialized form of hybridization. This technique is based on the use of Northern and Southern blotting techniques and requires the use of two nylon membranes of differing chemical characteristics. A nylon membrane containing permanently affixed, electrophoretically fractionated RNAs is contact-hybridized at a right angle to a second, chemically different nylon membrane containing transiently bound, fractionated labeled DNA fragments. RNA and DNA bands possessing homology will hybridize where they cross, forming an autoradiographically detectable spot. This Northern Cross procedure proportionately represents the amounts of different RNAs derived from a particular sequence in a manner similar to what would have been observed in a Northern blot. This method, which can be used in the analysis of even relatively rare RNA species, permits rapid and fairly inexpensive identification of exon-containing fragments or determination of the relationship between related, multiple RNA species.

Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis.

With the exception of the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum deltaH, all organisms surveyed contain orthologs of Escherichia coli cysteinyl-tRNA synthetase (CysRS). The characterization of CysRS-encoding (cysS) genes and the demonstration of their ability to complement an E. coli cysSts mutant reveal that Methanococcus maripaludis and Methanosarcina barkeri, two other methanogenic archaea, possess canonical CysRS proteins. A molecular phylogeny inferred from 40 CysRS sequences indicates that the CysRS of M. maripaludis and Methanosarcina spp. are specific relatives of the CysRS of Pyrococcus spp. and Chlamydia, respectively. This result suggests that the CysRS gene was acquired by lateral gene transfer in at least one euryarchaeotic lineage.

Nasal lavage as a tool in assessing acute inflammation in response to inhaled pollutants.

The upper airway, especially the nose, is a major target of toxic damage. Nasal challenges followed by nasal lavage (NAL) have been applied to studies of hypersensitivity, in particular as a method to identify the allergen in patients with allergic situations such as rhinitis. The NAL method has not been extensively used to determine the effects of air pollutants on the upper airways in humans. Ozone is known to interact avidly with various tissues in the respiratory tract and to cause decrements in lung function tests. This oxidant pollutant has also been shown to induce inflammation in the lower airways of humans and animals. In this study, we have examined the effect of an acute (2 h) exposure of ozone at 0.4 ppm on the inflammatory response in the upper airways of 10 normal volunteers and compared these results to those obtained in the lower airways assessed by bronchoalveolar lavage (BAL). The results indicate significant increases in the number of polymorphonuclear neutrophils (PMN) in NAL immediately post exposure (7.7-fold). This increase is still detectable 18 h post exposure (6-fold) which is similar to the increase of PMN in BAL. Tryptase, released by mast cells was also increased in the NAL fluid immediately post exposure (2-fold). While the albumin level, which is an indicator of epithelial cell permeability, was elevated 18 h post exposure (1.5-fold), tryptase level, was not anymore elevated at that time point. Interestingly, several other markers of acute inflammation such as prostaglandin E2 (PGE2), C3a, urokinase-type plasminogen activator (U-PA), which were found to be significantly elevated in the BAL of the same group of subjects (18 h post exposure), were not elevated in the NAL either immediately post or 18 h post exposure. The level of uric acid, thought to be an important anti-oxidant molecule, was also unchanged in the NAL fluid but was elevated in the BAL fluid. Collectively the data suggest that NAL may serve as a sensitive and reliable technique to detect inflammation in the upper airways of subjects exposed to ozone. Moreover, in the case of this particular oxidant pollutant, the NAL seems to mirror the inflammatory response in the lower airways, 18 h post exposure, relative to the number of PMN and albumin levels.

Exposure of humans to a volatile organic mixture. III. Inflammatory response.

A set of symptoms has been described during the past two decades that has been called the "sick building syndrome." These symptoms include eye, nose, and throat irritation; headache; mental fatigue; and respiratory distress. It is likely that the volatile organic compounds (VOCs) present in synthetic materials used in homes and office buildings contribute to these symptoms. However, there have been very few studies in which humans have been exposed to known amounts of VOCs under carefully controlled conditions. In this study, 14 subjects were exposed to a mixture of VOCs (25 mg/m3 total hydrocarbon) that is representative of what is found in new homes and office buildings. Because irritations of the nose and throat are symptoms often associated with the upper respiratory tract and may result from an inflammatory response in the upper airways, we used nasal lavage to monitor neutrophil (PMN) influx into the nasal passages following exposure to VOCs. There were statistically significant increases in PMNs, both immediately after a 4-h exposure to VOCs and 18 h later.

Profound ambulatory hypoglycemia: a rare entity.

A 49-year-old man with a history of hepatocellular carcinoma, alcohol abuse, and insulin-dependent diabetes mellitus was noted to be completely asymptomatic despite a plasma glucose level of 4 mg/dL. The possible pathophysiology of this unusual occurrence of "hypoglycemia unawareness" is discussed.

Quality and governance in hospitals.

What is the role of the board in quality initiatives in an organization? How can the board improve its own processes through quality initiatives? What are the quality attributes that a board should monitor? The board at St. Mary's Hospital in Kitchener addressed these questions, resulting in a rethinking of the board's role and its relationship to the operation of the hospital. This article discusses how the board has been restructured, and how it has shifted over the past few years to a CQI focus.

Interspersion of repetitive with repetitive sequences in an amphibian, Rana berlandieri.

When conventional genome arrangement analyses performed on R. berlandieri DNA at a normal (60 degrees C) and a high (75 degrees C) reassociation temperature were compared, an additional interspersion pattern was detected which indicates that different classes of repetitive sequences are closely interspersed with each other. Our results further suggest that the genomic abundance of purified (or cloned) repetitive sequences can be accurately determined by solution hybridization with genomic DNA only when the reassociation is performed at a relatively high temperature (Tm - 10 degrees C).

Biomarkers of inflammation in ozone-exposed humans. Comparison of the nasal and bronchoalveolar lavage.

Previously we established that an acute inflammatory response in the upper respiratory tract of humans could be studied by analyses of nasal lavages (NL). The relationship of these cellular responses to responses in the lower lung has not been thoroughly investigated in humans. In this study we have compared the cellular changes detected in NL with those detected in the bronchoalveolar lavage (BAL) taken from the same individual. A group of 10 subjects was exposed to either filtered air or 0.4 ppm ozone (O3), with exercise, for 2 h. The NL was done prior to, immediately following, and 18 h postexposure; the BAL was done only at 18 h postexposure. A significant increase in PMN was detected in the NL immediately postexposure to O3 (7.7-fold increase; p = 0.003) and remained elevated in the 18 h post-O3 NL (6.1-fold increase; p less than 0.001). A similar increase in PMN was detected in the BAL 18 h after exposure to O3 (6.0-fold increase; p less than 0.001). The albumin levels in the NL and BAL were also similarly increased 18 h after O3 (3.9-fold and 2.2-fold, respectively). Although a qualitative correlation in the mean number of PMN existed between the upper and lower respiratory tract after O3, comparison of the NL and BAL PMN from each individual showed a significant quantitative correlation for the air data (r = 0.741; p = 0.014) but not for the O3 data (r = 0.408; p = 0.243). This study demonstrates that PMN counts in the NL can be a useful, inexpensive means of studying the acute inflammatory effect of ozone and monitoring those effects in the lower lung.

Ion and sugar permeabilities of lecithin bilayers: comparison of curved and planar bilayers.

Na+ and sugar permeabilities of egg lecithin bilayers were measured using curved bilayers and planar bilayers as represented by single-bilayer vesicles and black lipid films, respectively. The Na+ permeability coefficient measured with single-bilayer vesicles at 25 degrees C is (2.1 +/- 0.6) x 10(-13) cm sec-1. Because of technical difficulties it has been impossible to measure ionic permeabilities of values lower than about 10(-10) cm sec-1 in planar (black) lipid bilayers using tracer methods. The D-glucose and D-fructose permeabilities were measured with both curved and planar bilayers. The permeability coefficients measured with vesicles at 25 degrees C are (0.3 +/- 0.2) x 10(-10) cm sec-1 for glucose and (4 +/- 1) x 10(-10) cm sec-1 for D-fructose; these are in reasonable agreement with the corresponding values obtained for planar (black) lipid bilayers which are (1.1 +/- 0.3) x 10(-10) cm sec-1 for D-fructose, respectively.

Increased concentration of an indigenous proviral mouse mammary tumor virus long terminal repeat-containing transcript is associated with neoplastic transformation of mammary epithelium in C3H/Sm mice.

Increased amounts of mouse mammary tumor virus (MMTV) proviral transcripts were found in RNA dot blots from MMTV-negative, C3H/Sm mouse mammary tumors which arose spontaneously or were induced by hormonal or chemical carcinogens or both. Other dot blots probed with a long terminal repeat (LTR) probe showed that LTR (MMTV)-containing transcripts were disproportionately represented in these tumor RNAs. Different segments of the MMTV genome were used in sequential hybridizations to Northern blots to determine relative sequence content and size of MMTV transcripts in transformed mammary tissues, as compared with those in lactating mammary glands. Increased amounts of 4.4-kilobase env and 8.1-kilobase genomic MMTV transcripts were detected with an env probe in many of the tumor RNAs examined. Hybridization of the same Northern blots containing tumor RNAs with an LTR probe revealed a 2.2-kilobase transcript which was prominent in RNAs from chemically-induced, hormonally-induced, and spontaneous mammary tumors relative to those from lactating mammary glands. The LTR-containing transcript did not possess significant homology to either env or gag-pol probes. This distinctive, transformation-enhanced, 2.2-kilobase transcript may contain mouse cellular sequences in addition to LTR sequences or it may represent the message for a nonstructural viral protein encoded within the LTR open reading frame of one or more of the four C3H/Sm MMTV proviral genes.