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PubMed1995

An endogenous lectin and its glycoprotein ligands are triggering basal and axon-induced Schwann cell proliferation.

The proliferation of Schwann cells (the myelinating cells of the peripheral nervous system) is stimulated by the contact with axonal membranes. It is suggested that the endogenous carbohydrate-binding protein (lectin) cerebellar soluble lectin (CSL) bound to ligands at the surface of axonal preparations is mitogenic for Schwann cells. Both autocrine and axon-stimulated Schwann cell proliferations seem to be dependent on the presence of CSL and its ligands at the Schwann cell surface, as suggested by the effects of N-glycosylation inhibitors and anti-CSL Fab fragments. These data suggest that CSL regulates Schwann cell proliferation by clustering of a few glycoprotein ligands at the cell surface, consequently modulating phosphorylations.

1-Deoxynojirimycin↗

PubMed1982

Automatic construction of restriction site maps.

A computer program is described which constructs maps of restriction endonuclease cleavage sites in DNA molecules, given only the fragment lengths. The program utilizes fragment length data from single and double restriction enzyme digests to generate maps for linear or circular molecules. The search for a map can be limited to the unknown (insert) region of a recombinant phage or plasmid. Typical restriction maps with four or five enzymes which cut at three to five unknown sites can be calculated in a few minutes.

Autoanalysis↗

PubMed1983

The coupled use of 'footprinting' and exonuclease III methodology for RNA polymerase binding and initiation. Application for the analysis of three tandem promoters at the control region of colicin El.

In order to determine the initiation site for three promoters P1, P2 and P3 (5' to 3') in close proximity in the colicin E1 control region we developed a new methodology that couples ternary complex formation and the analysis of the 3' border protected from exonuclease III digestion. The initiation of transcription could be detected by measuring the shift in the position of the 3' protected border when RNA polymerase moved from its binary complex position to its ternary complex position. The latter stops at a specific nucleotide because transcription is initiated with one or more NTPs missing. This approach, coupled with "footprinting", can also be used to decide whether the formation of an RNA polymerase binary or ternary complex at one site excludes or weakens binding at neighboring sites. The location of 3' protected borders reveals the formation of respective binary and ternary complexes at non-saturating RNA polymerase conditions, whereas at saturating conditions only the distal 3' boundary is seen and exonuclease cannot penetrate further. However, if "footprinting" reveals proximal 5' patterns this establishes that simultaneous binding has occurred on the same DNA fragment. The data showed that this was true for P1 and P3 which are only 8 nucleotides apart. P2 could only be detected at non-saturating conditions since it overlaps both P1 and P3. The evidence from the literature and this study establishes P1 as the true colicin E1 promoter with the possibility that supercoiling may eliminate any role for P2 and P3.

Amino Acid Sequence↗

PubMed1995

Specific inhibition of DNA polymerase beta by its 14 kDa domain: role of single- and double-stranded DNA binding and 5'-phosphate recognition.

DNA polymerase beta (beta-polymerase) has been implicated in short-patch DNA synthesis in the DNA repair pathway known as base excision repair. The native 39 kDa enzyme is organized into four structurally and functionally distinct domains. In an effort to examine this enzyme as a potential therapeutic target, we analyzed the effect of various beta-polymerase domains on the activity of the enzyme in vitro. We show that the 14 kDa N-terminal segment of beta-polymerase, which binds to both single- and double-stranded DNA, but lacks DNA polymerase activity, inhibits beta-polymerase activity in vitro. Most importantly, the 8, 27 and 31 kDa domains of beta-polymerase do not inhibit beta-polymerase activity, demonstrating that the inhibition by the 14 kDa domain is specific. The inhibition of beta-polymerase activity in vitro is abolished by increasing the concentrations of both of the substrates (template-primer and deoxynucleoside triphosphate). In contrast, an in vitro base excision repair assay is inhibited in a domain specific manner by the 14 kDa domain even in the presence of saturating substrates. The inhibition of beta-polymerase activity by the 14 kDa domain appears specific to beta-polymerase as this domain does not inhibit either mammalian DNA polymerase alpha or Escherichia coli polymerase I (Klenow fragment). These data suggest that the 14 kDa domain could be used as a potential inhibitor of intracellular beta-polymerase and that it may provide a means for sensitizing cells to therapeutically relevant DNA damaging agents.

Base Sequence↗

PubMed1996

Structure of the distal human gonadotropin releasing hormone (hGnrh) gene promoter and functional analysis in Gt1-7 neuronal cells.

To assess potential species-specific expression of gonadotropin releasing hormone (GnRH), the distal human (h) GnRH promoter was cloned, characterized and tested in gene transfer studies. The nucleotide sequence of approximately 3.8 kb of 5'-flanking region was determined. Homology to the rat (r) GnRH sequence was observed in the proximal promoter region between -551 h (-424 r) and the transcriptional start site and within multiple distal promoter regions. In contrast, there was little similarity in the sequences between -1131/-551 h and -1031/-424 r. A deletion panel of 5'-flanking hGnRH promoter constructs was made and tested in transient transfection assays in GnRH-producing mouse GT1-7 neuronal cells. The largest hGnRH promoter construct (-3832/+5 h) exhibited high levels of reporter activity, similar to that observed with the largest rGnRH construct (-3026/+116 r). However, in contrast to the rat gene, deletion of distal promoter sequences of the hGnRH promoter to -1971, -1131 or -551 did not result in a decrease in luciferase reporter activity. Further truncation to -350 resulted in a 3-fold decrease in luciferase activity. There was no preferential use of the putative upstream hGnRH start site in neuronal cells. DNase I protection assays showed unique protection patterns with nuclear extracts from GT1-7 and Gn10 neuronal cells and the hGnRH and rGnRH promoter fragments. These data suggest the presence of different cis-acting elements and transacting factors that mediate species-specific neuronal GnRH expression.

Animals↗

PubMed2001

GenMapDB: a database of mapped human BAC clones.

GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9-22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequences of the clones. GenMapDB is freely available to the public. The main purpose of GenMapDB is to organize the mapping data and to allow the research community to search for mapped BAC clones that can be used in gene mapping studies and chromosomal mutation analysis projects.

Chromosome Mapping↗

PubMed1981

The structure of a thirty-six kilobase region of the human chromosome including the fibroblast interferon gene IFN-beta.

The isolation of a human genomic cosmid hybrid containing the interferon beta gene has recently been reported (Gross et al., 1981). This hybrid was mapped using single and double digests and cross-hybridisation with the sub-cloned EcoRI and BgIII fragments. Purified fragments and subclones were used as hybridisation probes against chromosomal "Southern" blots to show that at least half of the region has been cloned without alteration. This cannot at present be confirmed for the rest of the region due to the presence of highly repetitive DNA on these cloned fragments. Sequencing data on the 5'-end of the fibroblast IFN-beta gene shows a high density of direct and inverted repeats. The IFN-beta mRNA coding region contains no intron, although the possibility of other transcription starts is not ruled out. The cloned region shows no similarities to known genomic clones containing IFN-alpha genes.

Base Sequence↗

PubMed2002

Effects of deletion of streptokinase residues 48-59 on plasminogen activation.

Streptokinase (SK) is a thrombolytic agent widely used for the clinical treatment of clotting disorders such as heart attack. The treatment is based on the ability of SK to bind plasminogen (Pg) or plasmin (Pm), forming complexes that proteolytically activate other Pg molecules to Pm, which carries out fibrinolysis. SK contains three major domains. The N-terminal domain, SKalpha, provides the complex with substrate recognition towards Pg. SKalpha contains a unique mobile loop, residues 45-70, absent in the corresponding domains of other bacterial Pg activators. To study the roles of this loop, we deleted 12 residues in this loop in both full-length SK and the SKalpha fragment. Kinetic data indicate that this loop participates in the recognition of substrate Pg, but does not function in the active site formation in the activator complex. Two crystal structures of the deletion mutant of SKalpha (SKalpha(delta)) complexed with the protease domain of Pg were determined. While the structure of SKalpha(delta) is essentially the same as this domain in full-length SK, the mode of SK-Pg interaction was however different from a previously observed structure. Even though mutagenesis studies indicated that the current complex represents a minor interacting form in solution, the binding to SKalpha(delta) triggered similar conformational changes in the Pg active site in both crystal forms.

Base Sequence↗

PubMed1986

Membrane glycoproteins from spermatozoa: partial characterization of an integral Mr = approximately 24,000 molecule from rat spermatozoa that is glycosylated during epididymal maturation.

Spermatozoa from the rat cauda epididymidis were treated with either the galactose oxidase-NaB[3H]4 or the NaIO4-NaB [3H]4 technique to label cell surface moieties of galactose and sialic acid, respectively. Following extraction with 40 mM octyl-beta-D-glucopyranoside (OBG), electrophoresis in sodium dodecylsulfate-polyacrylamide gels (SDS-PAGE) revealed a single radioactive peak migrating at Mr = approximately 24,000 in 11.2%, 14% and 16.8% tube gels. SDS-PAGE of the same OBG extract on 5.6% and 14% gels showed that this molecule was the same as that reported elsewhere, having a molecular weight varying from 32,000 to 37,000. The amount of labeled molecule extracted with 8 M urea or with 6 M guanidine-HCl was 30 and 50%, respectively, of that achieved with OBG. However, when labeled sperm were treated under other conditions (at pH 8, pH 3, 0.1-3 M NaCl [at pH 7.2], 5 mM ethylenediaminetetraacetic acid [EDTA], 20-200 mM dithiothreitol or 20-200 mM betamercaptoethanol, at varying temperatures and extraction times), the amount of labeled molecule extracted was less than 1% of that obtained with OBG. The molecule aggregates in aqueous buffers, as shown by chromatography using Sephadex G-100 and by centrifugation through a sucrose density gradient. Analysis by charge-shift electrophoresis suggested that the molecule contains an exposed hydrophobic domain(s). Mild trypsin treatment released all the labeled carbohydrate with the majority of the label attached to a Mr = 10,000 fragment. These data support the hypothesis that the molecule is an integral membrane glycoprotein, and suggest that it is only partially buried in the lipid matrix of the plasma membrane.

Animals↗

PubMed1992

Induction of ovulation and oocyte maturation of amphibian (Rana dybowskii) ovarian follicles by protein kinase C activation in vitro.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Intracellular degradation of the C-peptide of proinsulin, in a human insulinoma: identification of sites of cleavage and evidence for a role for cathepsin B.

An extract of a neuroendocrine tumor of the human pancreas contained a high concentration of insulin and the C-peptide of proinsulin, as determined by radioimmunoassay, together with somatostatin, calcitonin, and thymosin beta 4. Analysis of the molecular forms of the proinsulin-derived peptides by high-performance liquid chromatography demonstrated that insulin was stored in the tumor as the intact peptide. In contrast, metabolites of C-peptide, representing the (1-21), (1-23), (1-25) and (1-29) N-terminal fragments, were isolated from the extract in addition to intact C-peptide. Generation of these metabolites involves cleavage of Xaa-Leu or Leu-Xaa bonds. Previous immunohistochemical studies have identified cathepsin B in secretory granules and lysosomes of human insulinoma cells. Synthetic human C-peptide was rapidly cleaved by purified human cathepsin B, primarily at the site of leucine residues, to give several metabolites, including the (1-25) and (1-23) fragments. The data indicate that the C-peptide of proinsulin is selectively metabolized in the neoplastic B cell by a mechanism that involves proteolytic cleavages in the C-terminal region of the peptide.

Aged↗

PubMed1986

Specificity and precursor frequency of cytotoxic T cells in the response to minor H antigens.

The cytotoxic T cell response to minor histocompatibility antigens (MIHA) has been characterized in terms of the specificity repertoire, Lyt phenotype and precursor frequency. In the response of primed BALB/c spleen cells to B10.D2 or DBA/2 MIHA, the cytotoxic T lymphocyte (CTL) generated bear Thy-1.2, and are Lyt-1+2+. In the response to intact stimulator cells we have been able to distinguish at least three different specificities of anti-MIHA CTL in the response of primed BALB/c T cells to DBA/2 MIHA. These subsets exhibit differential inductive requirements and are derived from CTL precursors (CTLp) present at different frequencies in primed spleen. As reported by others, at high responder cell numbers (10(7)) the CTL generated see MIHA shared between DBA/2 and B10.D2 in H-2-restricted fashion. However, at lower numbers (3 X 10(5)), the CTL generated are only weakly crossreactive between DBA/2 and B10.D2 as defined by cold target competition. These CTL show a higher activity for DBA/2 than for B10.D2 regardless of the hot target used in the competition assay. CTLp are depleted by absorption on DBA/2 but not B10.D2 monolayers. These CTLp are present at higher frequency than are the CTLp responsive to shared MIHA--yet are undetectable at high responder cell numbers, suggesting strong clonal dominance effects. At low cell numbers (3 X 10(4] in the response to intact stimulators, the CTL are specific for MIHA unique to DBA/2, and crossreactive CTL able to lyse B10.D2 are no longer detectable. Addition of growth factors to cultures restores the crossreactive CTL, suggesting a requirement for higher levels of help and indicating that these two subsets derive from different CTLp. Finally the frequency of CTLp responsive to MIHA on intact cells was found to be 40-fold higher than the frequency of CTLp responsive to MIHA on membrane fragments. These data are discussed in terms of strong immunodominance mechanisms that appear to operate in the response to MIHA.

Animals↗

PubMed2003

Traumatic axonal injury results in biphasic calpain activation and retrograde transport impairment in mice.

Traumatic axonal injury (TAI) is one of the most important pathologies associated with closed head injury, and contributes to ensuing morbidity. The authors evaluated the potential role of calpains in TAI using a new model of optic nerve stretch injury in mice. Male C57BL/6 mice were anesthetized, surgically prepared, and subjected to a 2.0-mm optic nerve stretch injury (n = 34) or sham injury (n = 18). At various intervals up to 2 weeks after injury, optic nerves were examined for neurofilament proteins and calpain-mediated spectrin breakdown products using immunohistochemistry. In addition, fluorescent tracer was injected into the superior colliculi of mice 1 day before they were killed, to investigate the integrity of retrograde axonal transport to the retina. Optic nerve stretch injury resulted in persistent disruption of retrograde axonal transport by day 1, progressive accumulation and dephosphorylation of neurofilament protein in swollen and disconnected axons, and subsequent loss of neurofilament protein in degenerating axons at day 14. Calpains were transiently activated in intact axons in the first minutes to hours after stretch injury. A second stage of calpain-mediated proteolysis was observed at 4 days in axonal swellings, bulbs, and fragments. These data suggest that early calpain activation may contribute to progressive intraaxonal structural damage, whereas delayed calpain activation may be associated with axonal degeneration.

Animals↗

PubMed2000

Dispersal and extinction in fragmented landscapes.

Evolutionary and population dynamics models suggest that the migration rate will affect the probability of survival in fragmented landscapes. Using data for butterfly species in the fragmented British landscape and in immediately adjoining areas of the European continent, this paper shows that species of intermediate mobility have declined most, followed by those of low mobility, whereas high-mobility species are generally surviving well. Compared to the more sedentary species, species of intermediate mobility require relatively large areas where they breed at slightly lower local densities. Intermediate mobility species have probably fared badly through a combination of metapopulation (extinction and colonization) dynamics and the mortality of migrating individuals which fail to find new habitats in fragmented landscapes. Habitat fragmentation is likely to result in the non-random extinction of populations and species characterized by different levels of dispersal, although the details are likely to depend on the taxa, habitats and regions considered.

Analysis of Variance↗

PubMed1993

A novel cis-stimulatory element maps to the 5' portion of the human papillomavirus type 18 upstream regulatory region and is functionally dependent on a sequence-aberrant Sp1 binding site.

Gene expression of the cancer-associated human papillomavirus (HPV) type 18 is modulated by cis-regulatory elements located within the viral upstream regulatory region (URR). All cellular factors identified so far involved in viral gene control bind to the 3' portion of the HPV-18 URR. In contrast, very little is known about regulatory elements within the 5' portion of the URR. We therefore analysed this region of unknown function to delineate potential cis-regulatory elements contained therein. By utilizing transient expression assays, an 84 bp fragment could be identified in the 5' portion of the URR that exhibits orientation-independent cis-stimulatory activity in HeLa cervical carcinoma cells and primary human fibroblasts. Gel retardation assays and competition experiments indicated specific binding of cellular proteins to the 84 bp fragment. By functional dissection, a regulatory element with intrinsic cis-stimulatory activity could be mapped within the 84 bp fragment. Binding studies indicate that this cis-stimulatory element contains a sequence-aberrant Sp1 recognition site. Transient luciferase assays performed with mutated templates demonstrate that this Sp1 binding site behaves as a functional Sp1 element in vivo and is a main determinant of the cis-stimulatory activity exerted by the 84 bp fragment. These data show that the 5' portion of the HPV-18 URR contains cis-activating elements and indicate an important functional role for the cellular transcription factor Sp1 in their regulation.

Base Sequence↗

PubMed1992

Comparison of enterotoxins and haemolysins produced by methicillin-resistant (MRSA) and sensitive (MSSA) Staphylococcus aureus.

A collection of 201 isolates of Staphylococcus aureus was examined: 152 methicillin-sensitive S. aureus (MSSA) comprised 48 blood culture isolates (BC) and 58 isolates from routine diagnostic specimens (RD) from Glasgow Royal Infirmary (GRI), and 46 strains from nasal swabs of patients attending a general practitioner (GP); 49 isolates were of methicillin-resistant S. aureus (MRSA) from GRI. We have previously shown that the MRSA could be divided into two sub-groups on the basis of sensitivity or resistance to aminoglycoside antibiotics. Production of enterotoxins A, B, C and D, and alpha-, beta-, gamma- and delta- haemolysins was detected by reverse passive latex agglutination (RPLA) and agar overlay methods respectively: 60% of BC MSSA and a similar proportion of MSSA from other sources produced enterotoxin; 87% of aminoglycoside-sensitive MRSA produced enterotoxin (89% of these produced enterotoxin A alone) whereas only 27% of aminoglycoside-resistant MRSA were enterotoxin-positive, significantly less than either MSSA or aminoglycoside-sensitive MRSA. The proportion of haemolysin-producing isolates did not differ amongst the isolates of MSSA and MRSA; there was no difference in the distributions of haemolysins between aminoglycoside-sensitive and -resistant strains of MRSA. GP MSSA had higher and lower numbers of gamma- and delta-haemolysin producers respectively than other S. aureus isolates. alpha-Haemolysin producers were commoner amongst MRSA isolates, which were also more likely than MSSA isolates to produce several haemolysins. Differences in enterotoxin production between aminoglycoside-sensitive and -resistant MRSA isolates reflect subgroups previously defined by biotype, phage type, immunoblot and restriction enzyme fragmentation pattern data, and provide further evidence for the existence of two major MRSA clones in GRI.

Aminoglycosides↗

PubMed1998

Alterations in the cytoskeleton accompany aluminum-induced growth inhibition and morphological changes in primary roots of maize

Although Al is one of the major factors limiting crop production, the mechanisms of toxicity remain unknown. The growth inhibition and swelling of roots associated with Al exposure suggest that the cytoskeleton may be a target of Al toxicity. Using indirect immunofluorescence microscopy, microtubules and microfilaments in maize (Zea mays L.) roots were visualized and changes in their organization and stability correlated with the symptoms of Al toxicity. Growth studies showed that the site of Al toxicity was associated with the elongation zone. Within this region, Al resulted in a reorganization of microtubules in the inner cortex. However, the orientation of microtubules in the outer cortex and epidermis remained unchanged even after chronic symptoms of toxicity were manifest. Auxin-induced reorientation and cold-induced depolymerization of microtubules in the outer cortex were blocked by Al pretreatment. These results suggest that Al increased the stability of microtubules in these cells. The stabilizing effect of Al in the outer cortex coincided with growth inhibition. Reoriented microfilaments were also observed in Al-treated roots, and Al pretreatment minimized cytochalasin B-induced microfilament fragmentation. These data show that reorganization and stabilization of the cytoskeleton are closely associated with Al toxicity in maize roots.

Journal Article↗

PubMed1989

Oncogene amplification in squamous cell carcinoma of the oral cavity.

We have determined the prevalence of amplification of c-myc, N-myc, L-myc, H-ras, Ki-ras, and N-ras oncogenes in 23 cases of squamous cell carcinoma of the oral cavity, using Southern hybridization analysis of DNA extracted from the primary tumor tissues. Nick-translated oncogene probes and oncogene inserts labeled to high specific activities were used. We observed a 5- to 10-fold amplification of one or more of c-myc, N-myc, Ki-ras and N-ras oncogenes in 56% of the tumor tissue samples, with these oncogenes not being amplified in the peripheral blood cells of the same patients. L-myc and H-ras were not amplified in any of our samples. The oncogene amplifications seemed to be associated with advanced stages of squamous cell carcinomas, with the ras and myc family oncogenes being amplified in stages 3 and 4. Hybridization with N-myc detected an additional 2.3 kb EcoRI fragment, along with the normal 2.1 kb fragment. Our data also demonstrated amplification of multiple oncogenes in the same tumor tissue sample. About 60% of the samples with amplified oncogenes showed simultaneous amplification of 2 or more oncogenes. The results showing different oncogene amplifications in similar tumors, as well as multiple oncogene amplifications in the same tumor, suggest that these oncogenes may be alternatively or simultaneously activated in oral carcinogenesis.

Adult↗