Analysis of a spatially regulated phosphotyrosine phosphatase identifies tyrosine phosphorylation as a key regulatory pathway in Dictyostelium. We have cloned a Dictyostelium phosphotyrosine phosphatase (PTP1) with a catalytic domain showing approximately 38%-50% amino acid identity to those of other PTPs. PTP1 contains an approximately 99 amino acid insert and bacterially produced PTP1 possesses PTP activity. PTP1 is expressed at a very low level in vegetative cells, induced by 4 hr, and maximally expressed at the tight aggregate stage. PTP1-lacZ studies indicate that PTP1 is spatially localized to prestalk and anterior-like cell types. PTP1 gene disruptants show accelerated development, whereas strains overexpressing PTP1 to a high level fail to aggregate. Strains overexpressing moderate levels exhibit severe morphological defects following aggregation, including multiply tipped aggregates and morphologically aberrant fruiting bodies. Western blot analysis using anti-phosphotyrosine antibodies shows specific changes in the mutant strains when compared with wild-type cells. The results indicate that reversible protein-tyrosine phosphorylation and PTP1 play important regulatory roles during Dictyostelium development.


ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain. Protein-tyrosine kinases (PTKs) play an integral role in T cell activation. Stimulation of the T cell antigen receptor (TCR) results in tyrosine phosphorylation of a number of cellular substrates. One of these is the TCR zeta chain, which can mediate the transduction of extracellular stimuli into cellular effector functions. We have recently identified a 70 kd tyrosine phosphoprotein (ZAP-70) that associates with zeta and undergoes tyrosine phosphorylation following TCR stimulation. Here we report the isolation of a cDNA clone encoding ZAP-70. ZAP-70 represents a novel PTK and is expressed in T and natural killer cells. Moreover, tyrosine phosphorylation and association of ZAP-70 with zeta require the presence of src family PTKs and provide a potential mechanism by which the src family PTKs and ZAP-70 may interact to mediate TCR signal transduction.


The three-dimensional structure of the tenth type III module of fibronectin: an insight into RGD-mediated interactions. The solution structure of the tenth type III module of fibronectin has been determined using nuclear magnetic resonance techniques. The molecule has a fold similar to that of immunoglobulin domains, with seven beta strands forming two antiparallel beta sheets, which pack against each other. Both beta sheets contribute conserved hydrophobic residues to a compact core. The topology is more similar to that of domain 2 of CD4, PapD, and the extracellular domain of the human growth hormone receptor than to that of immunoglobulin C domains. The module contains an Arg-Gly-Asp sequence known to be involved in cell adhesion. This tripeptide is solvent exposed and lies on a conformationally mobile loop between strands F and G, consistent with its cell adhesion function.


RNA and DNA binding zinc fingers in Xenopus TFIIIA. The nine tandem zinc finger repeats in the 5S gene-specific transcription factor IIIA (TFIIIA) from Xenopus mediate specific binding to 5S DNA as well as to 5S ribosomal RNA. A comparative functional analysis of a systematic set of TFIIIA zinc finger combinations reveals that most, if not all, participate in both DNA and RNA binding. Minimal sets of fingers sufficient for DNA and RNA recognition are different. In RNA binding, most finger elements are found to be functionally equivalent. However, the nonessential finger 6 exhibits RNA binding characteristics distinct from the other eight modules. The secondary/tertiary structure of the central domain in 5S RNA, not its primary sequence, is found to carry the essential structural information for TFIIIA binding in Xenopus oocytes. Taken together, our findings suggest that RNA and DNA binding are overlapping, though separable functions of the nine zinc finger elements in TFIIIA, occurring via fundamentally different molecular mechanisms.


Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. We have identified a human homolog of the Drosophila trithorax protein that is structurally altered by 11q23 translocations in acute leukemias. Human trithorax (HRX) is a predicted 431 kd protein containing two potential DNA-binding motifs consisting of zinc fingers conserved with the fly protein and nonconserved amino-terminal "AT hook" motifs related to the DNA-binding motifs in HMG proteins. 11q23 translocations disrupt the HRX gene between these two motifs, and in a t(11;19)-carrying cell line fusion transcripts are expressed from both derivative chromosomes. The more abundant derivative 11 transcript codes for a chimeric protein containing the AT hook motifs fused to a previously undescribed protein (ENL) from chromosome 19. These data suggest a novel role for a trithorax-homologous protein in multilineage human leukemias that may be mediated by DNA binding within the minor groove at AT-rich sites, implicated to play an important role in bacterial IHF-, yeast datin-, and mammalian HMG-mediated gene activation.


The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. The ALL-1 gene located at human chromosome 11 band q23 is rearranged in acute leukemias with interstitial deletions or reciprocal translocations between this region and chromosomes 1, 4, 6, 9, 10, or 19. The gene spans approximately 100 kb of DNA and contains at least 21 exons. It encodes a protein of more than 3910 amino acids containing three regions with homology to sequences within the Drosophila trithorax gene, including cysteine-rich regions that can be folded into six zinc finger-like domains. The breakpoint cluster region within ALL-1 spans 8 kb and encompasses several small exons, most of which begin in the same phase of the open reading frame. The t(4;11) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-1 and from a gene on chromosome 4. This suggests that each 11q23 abnormality gives rise to a specific oncogenic fusion protein.


Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. The potent mesoderm-inducing factors activin and FGF are present as maternally synthesized proteins in embryos of X. laevis. We show that activin can act on explanted blastomeres to induce at least five different cell states ranging from posterolateral mesoderm to dorsoanterior organizer mesoderm. Each state is induced in a narrow dose range bounded by sharp thresholds. By contrast, FGF induces only posterolateral markers and does so over relatively broad dose ranges. FGF can modulate the actions of activin, potentiating them and broadening the threshold-bounded dose windows. Our results indicate that orthogonal gradients of activin and FGF would be sufficient to specify the main elements of the body plan.


Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. In MDCK cells, transport of membrane proteins to the basolateral plasma membrane has been shown to require a distinct cytoplasmic domain determinant. Although the determinant is often related to signals used for localization in clathrin-coated pits, inactivation of the coated pit domain in the human LDL receptor did not affect basolateral targeting. By expressing mutant and chimeric LDL receptors, we have now identified two independently acting signals that are individually sufficient for basolateral targeting. The two determinants mediate basolateral sorting with different efficiencies, but both contain tyrosine residues critical for activity. The first determinant was colinear with, but distinct from, the coated pit domain of the receptor. The second was found in the C-terminal region of the cytoplasmic domain of the receptor and, although tyrosine-dependent, did not mediate endocytosis. The results suggest that membrane proteins can have functionally redundant signals for basolateral transport and that a tyrosine-containing motif may be a common feature of multiple intracellular sorting events.


COP1, an Arabidopsis regulatory gene, encodes a protein with both a zinc-binding motif and a G beta homologous domain. Plant seedling development is capable of following 1 of 2 distinct morphogenic pathways: skotomorphogenesis in darkness and photomorphogenesis in light. Dark-grown Arabidopsis seedlings with recessive mutations at the constitutively photomorphogenic (COP1) locus indicate that the wild-type COP1 protein represses photomorphogenesis in darkness and that light reverses this repressive activity. Using a T-DNA-tagged mutant, we have cloned the COP1 locus. The amino-terminal half of the encoded protein contains a conserved zinc-binding motif, whereas the carboxyl-terminal half contains a domain homologous to the WD-40 repeat motif of G beta proteins. The presence of both a putative DNA-binding motif and a G protein-related domain in a single polypeptide suggests that COP1 may be the first of a new class of regulatory molecules. This novel structure could endow COP1 with the capacity to function as a negative transcriptional regulator capable of direct interaction with components of the G protein signaling pathway.


A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome. Prior to the chemical steps of mRNA splicing, the extensive base-pairing interaction between the U4 and U6 spliceosomal snRNAs is disrupted. Here, we use a mutational analysis in yeast to demonstrate a conserved base-pairing interaction between the U6 and U2 snRNAs that is mutually exclusive with the U4-U6 interaction. In this novel pairing, conserved sequences in U6 interact with a sequence in U2 that is immediately upstream of the branch point recognition region. Remarkably, the residues in U6 that can be consequently juxtaposed with the intron substrate include those that have been proposed previously to be catalytic. Both the first and second steps of splicing are inhibited when this base-paired structure is mutated. These observations, together with the high conservation of the U2-U6 structure, lead us to propose that it might be a component of the spliceosomal active site.


Mutational analysis of the yeast U2 snRNA suggests a structural similarity to the catalytic core of group I introns. We have used an in vitro reconstitution system to determine the effects of a large number of mutations in the highly conserved 5' terminal domain of the yeast U2 snRNA on pre-mRNA splicing. Whereas many mutations have little or no functional consequence, base substitutions in two regions were found to have drastic effects on pre-mRNA splicing. A previously unrecognized function for the U2 snRNA in the second step of splicing was found by alteration of the absolutely conserved sequence AGA upstream of the branch point recognition sequence. The effects of these mutations suggest the formation of a structure involving the U2 snRNA similar to the guanosine-binding site found in the catalytic core of group I introns.


Nucleosome disruption at the yeast PHO5 promoter upon PHO5 induction occurs in the absence of DNA replication. Activation of the PHO5 gene in S. cerevisiae by phosphate starvation was previously shown to be accompanied by the disappearance of four positioned nucleosomes from the promoter. To investigate the mechanism, we replaced the PHO80 gene, a negative regulator of PHO5, by a temperature-sensitive allele. As a consequence, PHO5 can be activated in the presence of phosphate by a temperature shift from 24 degrees C to 37 degrees C. Under these conditions, the promoter undergoes the same chromatin transition as in phosphate-starved cells. Disruption of the nucleosomes by the temperature shift also occurs when DNA replication is prevented. Nucleosomes re-form when the temperature is shifted from 37 degrees C back to 24 degrees C in nondividing cells. Glucose is required for the disruption of the nucleosomes during the temperature upshift, not for their re-formation during the temperature downshift. These experiments prove that DNA replication is not required for the transition between the nucleosomal and the non-nucleosomal state at the PHO5 promoter.


A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Tissue-specific patterns of methylated deoxycytidine residues in the mammalian genome are preserved by postreplicative methylation of newly synthesized DNA. DNA methyltransferase (MTase) is here shown to associate with replication foci during S phase but to display a diffuse nucleoplasmic distribution in non-S phase cells. Analysis of DNA MTase-beta-galactosidase fusion proteins has shown that association with replication foci is mediated by a novel targeting sequence located near the N-terminus of DNA MTase. This sequence has the properties expected of a targeting sequence in that it is not required for enzymatic activity, prevents proper targeting when deleted, and, when fused to beta-galactosidase, causes the fusion protein to associate with replication foci in a cell cycle-dependent manner.


Regulation of the specific DNA binding function of p53. The DNA binding activity of p53 is required for its tumor suppressor function; we show here that this activity is cryptic but can be activated by cellular factors acting on a C-terminal regulatory domain of p53. A gel mobility shift assay demonstrated that recombinant wild-type human p53 binds DNA sequence specifically only weakly, but a monoclonal antibody binding near the C terminus activated the cryptic DNA binding activity stoichiometrically. p53 DNA binding could be activated by a C-terminal deletion of p53, mild proteolysis of full-length p53, E. coli dnaK (which disrupts protein-protein complexes), or casein kinase II (and coincident phosphorylation of a C-terminal site on p53). Activation of p53 DNA binding may be critical in regulation of its ability to arrest cell growth and thus its tumor suppressor function.


The impairment of natural killer function in the healthy aged is due to a postbinding deficient mechanism. In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolytic activity which occurs during human aging, we examined by multiparametric flow cytometry the binding and lytic activities of human natural killer cells. The flow analysis revealed a striking increase of the CD16+8- subset, together with a significant decrease of CD8bright cells and total T cells (CD3+). Aging had no influence on the CD8dim subset. The total lytic activity expressed by PBL as well as their binding efficiency to K562 targets were moderately but not significantly increased in the elderly. In contrast, the cytotoxicity of the single target-bound natural killer cell (i.e., lytic efficiency) was deeply impaired in aged subjects, suggesting that the NK functional impairment observed in aging is located at postbinding level.


Synergistic enhancement of T cell responses and interleukin-1 receptor expression by interleukin-1 and heparin or dextran sulfate. Heparin markedly enhances generation of cytotoxic T lymphocytes against allogeneic cells and histocompatible tumors. In this study, we demonstrated a marked synergism between heparin and low concentrations of recombinant IL-1-alpha and IL-1-beta in enhancement of cytotoxic T cell responses in mice. Low molecular weight (8000 Da) dextran sulfate also enhanced the T cell responses and synergized with IL-1, whereas, de-N-sulfated heparin was devoid of both of these activities. The synergistic effect was selective for IL-1, because there was no synergism between heparin or dextran sulfate and other cytokines (tumor necrosis factor-alpha, IL-4, and, as shown previously, IL-2). Heparin did not increase the production of IL-1 (and IL-2, as shown before). Heparin did not bind to IL-1, despite significant amino acid homology between IL-1 and heparin-binding endothelial cell growth factors. Heparin enhanced the growth-promoting effect of IL-1 on the IL-1-dependent helper T cell clone, D10.G4.1, and enhanced IL-1 receptor expression on these cells. These data indicate that heparin acts directly on the T cells and enhances their responsiveness to IL-1 by up-regulating IL-1 receptor expression.


Abnormal activation and loss of suppressor T cells in the spontaneously hypertensive rat. Suppressor T cell function in the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rats was analyzed using syngeneic mixed lymphocyte reaction (SMLR) and concanavalin A (Con A) activation. A depressed SMLR was found in adult SHR but not in adult WKY. IL-2 synthesized by SHR was 40-fold lower than that of WKY, and the suppressor T cells generated in the SMLR were incapable of suppressing IgG synthesis. Precursors of cells that can be activated by Con A to become functional suppressor cells are reduced in adult SHR. Supernatant fluids derived from Con A-activated spleen cells from adult SHR failed to significantly inhibit IgG synthesis by cultures of syngeneic spleen cells compared to supernatant fluids from young SHR or WKY Con A-activated spleen cells. However, spleen cells from both adult SHR and WKY proliferated strongly and released equivalent amounts of IL-2 in response to Con A. Addition of exogenous IL-2 to the SMLR cultures in vitro restored the ability of SHR T cells to respond in the SMLR, with generation of cells capable of suppressing IgG synthesis. Administration of SHR with IL-2 in vivo also restored the suppressor T cell function in the SMLR. These results suggest a defective suppressor T cell activation and loss of suppressor T cell activity as the SHR age.


Stress stimuli-induced lymphocyte activation. Oxidants, heavy metals, and heat shock, collectively known as stress stimuli, induce the synthesis of a variety of proteins, termed stress proteins, and enhance glucose uptake. In this study, we have demonstrated that stress stimuli enhance protein tyrosine phosphorylation (PTyr-P), modulate protein tyrosine phosphatase (PTPase) activity, activate the src family protein tyrosine kinase (PTK), p56lck, and enhance glucose uptake in human peripheral blood mononuclear cells. The heavy metal Hg2+ and heat shock stimulated PTPase activity at an optimal dose, whereas the oxidant phenylarsine oxide (PAO) was only marginally stimulatory. Treatment of lymphocytes with stress stimuli at a dose which activated PTPase did not produce discernable PTyr-P using Western blotting techniques. PTyr-P was only seen at doses of stress stimuli which were associated with an inhibition of PTPase activity. We could demonstrate a correlation between the dose of stress stimuli effective in increasing PTPase activity and p56lck activation using heat shock and Hg2+ as stress stimuli. On the other hand, much lower concentrations of PAO were effective in activating PTPase than those effective in eliciting p56lck activation. We could not demonstrate a correlation between an effective dose inducing PTyr-P and glucose uptake. Our data do not permit us to draw a simple correlation between enhancement of PTPase activity, activation of p56lck, induction of PTyr-P, and induction of the biological response. It is possible that both stimulation and inhibition of PTPase could regulate PTyr-P by either activating the src family PTKs or preventing dephosphorylation of target proteins which are involved in the biological response. Our data may also provide the biochemical basis for the previously reported mitogenic effects of Hg2+ on lymphocytes.


IL-1, IL-6, and PDGF mRNA expression in alveolar cells following stimulation with a tobacco-derived antigen. To test the hypothesis that inflammatory cytokine production might be an early event in the development of the disease associated with smoking, we used alveolar cells from healthy nonsmokers stimulated with TGP as a model system. TGP, a phenol-rich glycoprotein which is present in tobacco leaves and cigarette smoke condensate, activates the immune system. It stimulates polyclonal B cell differentiation, induces primarily an IgE response, and activates human leukocytes to produce IL-1. Using in situ nucleic acid hybridization we show that the steady-state levels of IL-1 alpha, IL-1 beta, IL-6, platelet-derived growth factor (PDGF)-A, and PDGF-B mRNAs are consistently elevated in the alveolar cells of all donors following TGP stimulation. The kinetics of mRNA expression suggest that IL-1 alpha and IL-1 beta mRNAs are independently regulated in alveolar cells, while the regulation of PDGF-A and PDGF-B mRNA seems to be similar. The activated cells also synthesize elevated levels of IL-1 and IL-6. These findings lend support to the suggestion that some clinical consequences of smoking might be initiated and enhanced by the production of inflammatory cytokines. Moreover, IL-6 could also activate a polyclonal B cell response, which could lead to the synthesis of autoantibodies and thus cause immune-mediated tissue injury.


The CD45RO (p180, UCHL1) marker: complexity of expression in peripheral blood. Analysis of peripheral blood lymphocytes by two- and three-colour high-sensitivity staining with UCHL1 (CD45RO) and other markers shows that the expression of CD45RO on lymphocyte subsets is more complex than is generally supposed. In addition to the populations which express CD45RO and RA in a mutually exclusive manner, up to 30% of cells in adult blood express both markers, at low levels. This "intermediate" population includes CD4-positive cells, and a proportion of these cells express the p55 chain of the IL-2 receptor (CD25), suggesting that they are activated. In cord blood there are few RO-bright cells, but CD45RO is expressed at low intensity on a proportion of cells. Among the CD45RO-bright cells in adult blood at least two subsets can be detected by using MHC Class II and the homing receptor L-selectin as additional markers. This complexity suggests that memory cells are a subset of CD45RO-expressing cells, but that this marker is also found on cells that are activated but not irreversibly "switched" to memory cells.


Glutathione influences the proliferation as well as the extent of mitochondrial activation in rat splenocytes. The time-dependent changes of mitochondrial membrane potential and mass have been investigated on rat splenic lymphocytes stimulated with Con A in the presence and absence of reduced glutathione (GSH). Rhodamine-123 (Rh-123) and nonyl acridine orange (NAO) were used as specific dyes to monitor the membrane potential and mass of mitochondria, respectively. The percentage of cells showing blast transformation and the level of Rh-123 or NAO uptake were analyzed by flow cytometry. Present results demonstrate that a large number of cells showed activated mitochondria already at 24 hr after Con A stimulation and the activation of these organelles was not related to blast transformation. The addition of GSH into the culture medium increased the number of cells responding to mitogenic stimulation. In parallel it augmented the percentage of lymphocytes with activated mitochondria and also prevented their depolarization.


Tight conjugate formation is not always required for natural killer cell-mediated lysis. We have utilized several clonal cell lines, derived from the murine lymphoma ASL1w, to investigate the early events in NK-mediated lysis. The studies described here examine the relationship between NK recognition, NK cell:tumor cell conjugate formation, and NK-mediated lysis. The AW4F and AW4D tumor lines were susceptible to NK-mediated lysis and efficiently inhibited NK recognition in competitive inhibition assays, whereas the AW5J tumor, which is relatively resistant to NK-mediated lysis, did not. In contrast, the AW5E tumor was NK resistant but inhibited NK recognition almost as well as the NK-sensitive tumors, suggesting that it was deficient in a postbinding event required for NK-mediated lysis. These findings demonstrate a correlation, with one exception, between the susceptibility of the ASL1w-derived tumor lines to NK-mediated lysis and their ability to inhibit NK recognition. In contrast, there was no apparent correlation between tight conjugate formation, as assessed in three independent target binding assays, and the susceptibility of these tumors to NK-mediated lysis, showing that tight conjugate formation is not required for either efficient NK recognition or lysis.


Introduction of the H-2Dk gene into a class I-negative tumor cell line confers interferon-gamma inducibility upon the silent endogenous H-2Kk gene. Kgv cells do not constitutively express class I mRNA or protein. Interferon (IFN)-gamma, but not IFN-alpha/beta, induces H-2Dk expression. IFN does not induce H-2Kk expression. We examined constitutive and IFN-inducible class I expression on Kgv cells stably transfected with genomic clones of H-2Kk or H-2Dk and on somatic cell hybrid lines constructed between Kgv cells and constitutively class I-positive cells of a distinguishable H-2 haplotype. Our results suggest that both the lack of constitutive class I expression and the inability of IFN-alpha/beta to induce class I expression on Kgv cells are primarily due to cis-regulatory mechanisms. However, stable introduction of the H-2Dk gene into Kgv cells conferred IFN-gamma inducibility upon the silent endogenous H-2Kk gene. Therefore, the failure of IFN-gamma to induce H-2Kk expression on Kgv cells is due, at least in part, to a trans-regulatory mechanism.


Reduced binding activity of transcription factor NF-kappa B accounts for MHC class I repression in adenovirus type 12 E 1-transformed cells. The early region 1 (E1) of human adenovirus (Ad) type 12 represses the expression of major histocompatibility (MHC) Class I genes in transformed primary rodent cells. In this paper we show that both NF-kappa B and KBF1 (p50 dimer) binding activity to the H2TF1 element in the Class I promoter is reduced in Ad12-13S-E1A-transformed cells compared to Ad5E1- or Ad12-12S-E1A-transformed cells. Consistently, in Ad12E1A-13S-transformed cells the H2TF1 element does not contribute to transcriptional activity in transient expression assays, whereas it does contribute in Ad12E1A-12S-transformed cells. Therefore, the most likely explanation is that reduced binding of NF-kappa B and KBF1 to the H2TF1 element accounts for the down-regulation of MHC Class I expression in Ad12E1- and Ad12E1A-13S-transformed cells.


Generation of cytotoxicity with various MHC class I restrictions against autologous LCL by stimulating the lymphocytes with autologous and/or allogeneic LCLs sharing HLA alleles with the responder. We exposed human blood lymphocytes to autologous and to allogeneic lymphoblastoid lines (LCLs), each alone or in combination, and analyzed the MHC Class I restriction pattern of the generated auto-LCL reactive cytotoxicity. In the cultures of two EBV-seropositive, HLA A11-positive individuals the majority of cytotoxic lymphocytes generated after repeated stimulation with autologous LCL were restricted by this molecule. One of the cultures was subjected to various stimulation strategies. A relatively low proportion of HLA A2- and HLA B7-restricted cytotoxic T cells could be detected in the autostimulated cultures. Such cells were enriched at the expense of A11-restricted ones by stimulating with allogeneic LCLs which lacked HLA A11 but expressed A2 or B7. Interestingly, stimulation of the lymphocytes with only allogeneic LCL also generated autoreactive CTLs. Thus, by including or using exclusively allogeneic LCL stimulators, the CTL fractions represented by few cells could be enriched.


Relative resistance of intermediate TCR cells to anti-CD3 mAb in mice in vivo and their partial functional characterization. In addition to T cells differentiated in the thymus, T cells, possibly of extrathymic origin, were recently demonstrated in the liver of mice. These T cells are characterized by the expression of intermediate TCR and contain double-negative CD4-8- cells. A further characterization of intermediate TCR cells was carried out. When mice were injected ip with anti-CD3 mAb, bright TCR cells (i.e., regular T cells) and intermediate TCR cells were reduced on Day 3, depending on the amount of mAb. Because of the resistance of intermediate TCR cells to treatment, injection of an appropriate dose of antibody (i.e., 100 micrograms/mouse) eliminated most bright TCR cells, but not intermediate TCR cells. This dose revealed that a significant proportion of intermediate TCR cells also reside in the periphery. Hepatic and splenic mononuclear cells (MNC), in which intermediate TCR cells became abundant after treatment, showed a unique response to T cell mitogens and IL-2. Thus, the intermediate TCR cell-enriched population could not respond to a T cell mitogen, Con A, but responded well to a super antigen, staphylococcal enterotoxin B, and IL-2. MNC obtained from athymic nude mice, which comprise only intermediate TCR cells, responded in the same manner. These findings revealed that intermediate TCR cells are present not only in the liver but also in the periphery, and that they have a unique function distinct from regular T cells.


Microbiological and biotechnological aspects of metabolism of carbamates and organophosphates. Several carbamate and organophosphate compounds are used to control a wide variety of insect pests, weeds, and disease-transmitting vectors. These chemicals were introduced to replace the recalcitrant and hazardous chlorinated pesticides. Although newly introduced pesticides were considered to be biodegradable, some of them are highly toxic and their residues are found in certain environments. In addition, degradation of some of the carbamates generates metabolites that are also toxic. In general, hydrolysis of the carbamate and organophosphates yields less toxic metabolites compared with the metabolites produced from oxidation. Although microorganisms capable of degrading many of these pesticides have been isolated, knowledge about the biochemical pathways and respective genes involved in the degradation is sparse. Recently, a great deal of interest in the mechanisms of biodegradation of carbamate and organophosphate compounds has been shown because (1) an efficient mineralization of the pesticides used for insect control could eliminate the problems of environmental pollution, (2) a balance between degradation and efficacy of pesticides could result in safer application and effective insect control, and (3) knowledge about the mechanisms of biodegradation could help to deal with situations leading to the generation of toxic metabolites and bioremediation of polluted environments. In addition, advances in genetic engineering and biotechnology offer great potential to exploit the degradative properties of microorganisms in order to develop bioremediation strategies and novel applications such as development of economic plants tolerant to herbicides. In this review, recent advances in the biochemical and genetic aspects of microbial degradation of carbamate and organophosphates are discussed and areas in need of further investigation identified.


Protein engineering of antibodies.

Protein engineering of antibodies. This article reviews the technical advances in antibody engineering and the clinical applications of these molecules. Recombinant DNA technology facilitates the construction and expression of engineered antibodies. These novel molecules are designed to meet specific applications. Although genomic and cDNA cloning have been used widely in the past to isolate the relevant antibody V domains, at present, the PCR-based cloning is the preferred system. Bacterial and mammalian expression systems are used commonly for the production of antibodies, antibody fragments, and antibody fusion proteins. A range of chimeric antibodies with murine V domains joined to C regions from human and other species have been produced and found to exhibit the expected binding characteristics and effector functions. Humanized antibodies have been developed to minimize the HAMA response, and bifunctional immunoglobulins are being used in tumor therapy and diagnosis. Single chain antibodies and fusion proteins with antibody specificities jointed to nonimmunoglobulin sequences provide a source of antibody-like molecules with novel properties. The potential applications of minimal recognition units and antigenized antibodies are described. Combinatorial libraries produced in bacteriophage present an alternative to hybridomas for the production of antibodies with the desired antigen binding specificities. Future developments in this field are discussed also.


The modulation of collagen synthesis in cultured arterial smooth muscle cells by platelet-derived growth factor. Changes on collagen synthetic activity of cultured arterial smooth muscle cells of rabbits induced with purified platelet-derived growth factor (PDGF) were examined. PDGF treatment (final concentration was 5 units/ml) decreased the total collagen synthesis per cell, while the rate of collagen synthesis against total protein synthesis was raised by PDGF. Type analysis of collagen revealed substantial reduction of type IV collagen and relative increase of type V collagen in the PDGF-treated cells. By immunofluorescence study using anti-type IV collagen antibody, the lacework fluorescence was decreased with PDGF supplement. These findings indicate that PDGF induces the decrease of type IV collagen synthesis with the simultaneous diminution of basement membrane formation probably in association with phenotypic modulation of smooth muscle cells.


Subcellular distribution of hydralazine in rat single vascular muscle cells. High specific activity (20 Ci/mmol) tritiated hydralazine (3Hyd) distribution in isolated, cultured vascular muscle cells was determined to identify the sites of Hyd binding. 3Hyd dose-dependently bound to extracellular protein and to the area of organelles which secrete these proteins. Increased extracellular binding after Hyd pre-exposure suggests new binding sites may be exacerbated as a result of Hyd interactions. These experiments suggest a potentially important feature of the mechanism of action of this directly acting vasodilator.


A low molecular weight calf pineal peptide with an inhibiting effect on the growth of L1210 and HL60 cells. The peptide isolated by us from calf pineal gland causes a reduction of RNA synthesis in vitro in L1210 and HL60 tumoral cells. This peptide also causes inhibition of cell proliferation; the cell viability is not modified. The effects are dose-dependent and reversible.


Proliferation of the MAC-T bovine mammary epithelial cell line in the presence of mammary secretion whey proteins. Development of the MAC-T bovine mammary epithelial cell line by stable transfection with simian virus-40 large T-antigen should greatly assist study of possible intrinsic (local) and extrinsic (systemic) factors regulating bovine mammary epithelial cell development, differentiation, and function. This study evaluated the influence of mammary secretion whey proteins alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), lactoferrin (LF), transferrin (TF) and serum albumin (SA) on MAC-T cell proliferation in the absence and presence of 10% fetal bovine serum (FBS). Concentration of whey proteins in culture ranged from 0 to 625 micrograms/ml. MAC-T cell proliferation in the absence of FBS was significantly lower than in the presence of 10% FBS. Alpha-lactalbumin and LF significantly decreased MAC-T proliferation in both the absence and presence of 10% FBS. Transferrin significantly increased MAC-T cell proliferation only in the absence of FBS. There were no significant differences in MAC-T cell proliferation cultured in the presence of BLG or SA. These experiments illustrate the potential usefulness of MAC-T cells for the study of factors involved in mammary cell proliferation. Results identified ALA, LF and TF as possible intrinsic factors associated with regulation of mammary epithelial cell proliferation.


Simple method for isolation of primordial germ cells from chick embryos. A simple one step centrifugation method was developed for purification of primordial germ cells (PGCs) of chick embryos. PGCs, constituting less than 0.1% of the total blood cells of stage 13-14 embryos that contained a microliter amount of blood, were concentrated at the interface of a 6.3% (w/v) and 14.4% (w/v) Ficoll bilayer by centrifugation at 800 x g for 30 min. the purity of these PGCs was 86%, which was 22 times that obtained previously.


De novo biosynthesis and localization of immunoreactive inhibin (10.7 kDa) in normal human stomach. We have previously reported the occurrence of inhibin-like peptide in gastric juice of normal men. In the present investigation, normal gastric mucosa was shown to synthesize inhibin, in vitro, as measured by 3H-leucine incorporation (Maximum at 18 h). Furthermore, the immunohistochemical localization studies demonstrated its presence in the acid secreting parietal cells and basal region of foveolar epithelium of gastric mucosa. Surprisingly, the protein secreting zymogen cells remained unstained.


The involvement of Ca2+ and myosin light chain kinase in collagen-induced platelet activation. In this study we have used several complementary biochemical and immunological techniques to examine the involvement of Ca2+ and myosin light chain kinase in collagen-induced platelet activation. Our results indicate that collagen stimulates a rapid influx of external Ca2+ (within the first 1-5 min of treatment) which is followed by phosphorylation of myosin light chains (within 10 min of treatment) and granule secretion (within 15 min of treatment). In addition, we have found that certain Ca2+ channel entry blockers (e.g. nifedipine and bepridil) or calmodulin antagonists (e.g. W-7) specifically inhibit collagen-induced Ca2+ influx, myosin light chain phosphorylation and subsequent granule secretion. These data suggest that Ca2+/calmodulin-dependent myosin light chain kinase-mediated myosin light chain phosphorylation is necessary for regulating the actomyosin-related contractility required for normal platelet function.


The effects of staurosporine and okadaic acid on baby hamster kidney fibroblast cell adhesion. The effects of staurosporine, a potent protein kinase C inhibitor, and okadaic acid, a non-TPA tumour promoter, on the adhesion of BHK fibroblast were investigated. Staurosporine at 2.5 and 5 microM was found to stimulate a gradual increase in BHK cell adhesion as well as spreading in 3% serum-containing medium. An increase of approximately 27% over the control value was found at 5 microM concentration in 20 minutes. No such effect was seen in serum-free conditions. Staurosporine at 5 microM, enhanced BHK cell-cell adhesion in 3% serum and in serum-free conditions. Okadaic acid, a phosphatase inhibitor, at concentrations between 0.25 and 1 microgram/ml, was found to inhibit BHK cell-substratum adhesion and spreading. The inhibitory effect was time and concentration dependent. These findings suggest that protein kinase C might be involved in the mechanism(s) controlling BHK cell attachment.


TGF-beta is a potent inhibitor of hepatocyte growth factor secretion by human fibroblasts. Transforming growth factor-beta 1 (TGF-beta 1) inhibited secretion of human hepatocyte growth factor (hHGF), which is also known as scatter factor or fibroblast-derived tumor cytotoxic factor, by MRC-5 cells. The effect was detectable at as little as 10 pg/ml and was more potent than that of dexamethasone. Complete inhibition was observed after 12 h in the presence of 5 ng/ml of TGF-beta 1. Phorbol 12-myristate 13-acetate-induced secretion of hHGF from human skin fibroblasts was also suppressed by TGF-beta 1. TGF-beta 2 inhibited hHGF secretion by MRC-5 cells to the same extent as TGF-beta 1, but other growth factors such as epidermal growth factor and acidic and basic fibroblast growth factors had only a slight or null inhibitory effect.


The ERE-like sequence from the promoter region of the testis specific hsp70-related gene is not estrogen responsive. The 5'flanking region of the hsp70-related gene which is specifically expressed in rat and mouse spermatocytes contains the GGTCAnnnCGACC sequence closely resembling the palindromic consensus ERE (estrogen responsive element, GGTCAnnnTGACC). We used a transient expression assay to study whether this ERE-like sequence can transfer estrogen responsiveness either to heterologous or homologous promoters linked to a reporter CAT gene. We found that the GGTCAnnnCGACC sequence either as an isolated element or in its natural sequence context does not respond to hormone stimulation in MCF-7 and Fe33 cells. This observation points to the importance of the T-nucleotide in a right half-palindrome of ERE for ligand-dependent activation of transcription.


Chromosome mal-orientation and reorientation during mitosis. We argue that mal-orientation of mitotic chromosomes is not as rare as once believed. However, unlike bivalents during meiosis I, the reorientation of a mal-oriented mitotic chromosome has yet to be observed. This appears to be due, in part, to the difficulty in differentiating mal-oriented chromosomes from mono-oriented ones which are common during spindle formation in living mitotic cells. We assume that mitotic cells possess mechanisms for correcting chromosome mal-orientations that are similar to those operating during meiosis. However, unlike meiosis, where reorientation appears to be triggered when tension on a K-fiber is relieved or reduced, other factors related to the close proximity of sister kinetochores may also induce reorientation in mal-oriented mitotic chromosomes. We favor a model in which the reorientation of a mitotic kinetochore depends on, and is initiated by, the kinetochore capturing MTs from the pole to which it is reorienting.


Extension of filopodia by motor-dependent actin assembly. A variety of mechanisms have been proposed to explain the forward extension of cytoplasm in advancing cells and axonal growth cones, including actin polymerization and osmotic swelling. Based on our observations of the filopodia of cultured neuronal growth cones, we propose a mechanism involving motor-induced extension and retraction. We observed that filopodia (actin-based protrusions 0.2-0.5 mu in diameter) extend and retract from growth cone lamellae at the same rate. Further, force is generated at the tips of filopodia which is sufficient to produce compressive buckling of the proximal portion of the filopodium. From our analysis of these movements we suggest that a motor protein powers both the extension and retraction of filopodia.


Amino acid alterations in the benA (beta-tubulin) gene of Aspergillus nidulans that confer benomyl resistance. We report the cloning and sequencing of 18 mutant alleles of the benA, beta-tubulin gene of Aspergillus nidulans that confer resistance to the benzimidazole antifungal, antimicrotubule compounds benomyl, carbendazim, nocodazole, and thiabendazole. In 12 cases, amino acid 6 was changed from histidine to tyrosine or leucine. In four cases, amino acid 198 was changed from glutamic acid to aspartic acid, glutamine, or lysine. In two cases, amino acid 200 was altered from phenylalanine to tyrosine. These data, along with previous data indicating that amino acid 165 is involved in the binding of the R2 group of these compounds [Jung and Oakley, 1990: Cell Motil. Cytoskeleton 17:87-94], suggest that regions of beta-tubulin containing amino acids 6, 165, and 198-200 interact to form the binding site of benzimidazole antimicrotubule agents. These results also suggest that the presence of phenylalanine at amino acid 200 contributes to the great sensitivity of many fungi to benzimidazole antimicrotubule agents.


Colchicine-sensitive and colchicine-insensitive intermediate filament systems distinguished by a new intermediate filament-associated protein, IFAP-70/280 kD. A monoclonal antibody was produced, using as antigen a BHK-21 cytoskeletal preparation enriched in intermediate filaments (IF) and their associated proteins. This antibody reacted exclusively with a reproducible set of 70-280 kD polypeptides present in minor quantities in this preparation, as detected by immunoblot analysis. Based upon several criteria, this immunologically related group of polypeptides was designated as IFAP-70/280 kD (IF-Associated Protein): (1) it co-isolated with IF in vitro, (2) it co-localized (by both immunofluorescence and immunoelectron microscopy) with IF in situ in all stages of cell spreading, and (3) it segregated in vitro with the 54/55 kD (desmin/vimentin) structural IF subunit proteins of BHK cells through two cycles of in vitro disassembly/assembly. Immunogold labeling further localized IFAP-70/280 kD to regions of parallel or loosely bundled IF in situ, suggesting a role in regulating the supramolecular organization of IF. When this monoclonal antibody was used for double-label immunofluorescence observations of colchicine-treated BHK cells, it demonstrated the presence of colchicine-sensitive and colchicine-insensitive IF. Anti-IFAP-70/280 kD localized entirely to the drug-induced juxtanuclear IF cap, while a polyclonal antibody directed against the desmin/vimentin structural IF subunits and the previously characterized monoclonal anti-IFAP-300 kD [Yang et al., 1985; J. Cell Biol. 100:620] localized to both the juxtanuclear IF cap and a colchicine-insensitive IF network peripheral to the cap in the same cells. The colchicine-insensitive IF pattern often exhibited similarities to that observed for the actin-based stress fiber system, suggesting that stress fiber association may be an additional factor in IF organization.


Ca(2+)-regulated actin and phospholipid binding protein (68 kD-protein) from bovine liver: identification as a homologue for annexin VI and intracellular localization. An F-actin binding protein was purified from bovine liver by means of DNase I affinity, hydroxylapatite and DEAE-cellulose column chromatographies. It consisted of a single polypeptide chain having an apparent molecular weight of 68,000 with a Stokes radius of 35 A. Electron microscopy of rotary shadowed specimens showed that the 68 kD protein is a globular protein. This protein showed a higher affinity for F-actin in the presence of Ca2+ than in its absence, which is opposite to the actin-binding property shown by nonmuscle alpha-actinin or fimbrin. The 68 kD protein had no F-actin severing and capping activity. Interestingly, the 68 kD protein was found to aggregate liposomes at micromolar Ca2+ concentrations. Immunoblot analysis and partial protein sequence data identified the 68 kD protein as an annexin VI (p68) homologue. Immunocytochemical studies showed that the 68 kD protein was localized along stress fibers as well as membrane ruffles, microspikes and focal contacts, raising the possibility that annexin VI may contribute to control membrane-microfilament interaction in the cell.


Three-dimensional motility cycle in leukocytes. A 3-dimensional dynamic image analyzing system (3D-DIAS) has been developed in which a translocating cell is optically sectioned in the z-axis within a 2 sec period; the perimeter of the cell in each section is digitized into the 3D-DIAS data file, and the digitized perimeters are wrapped in order to reconstruct the cell image in three dimensions. Using 3D-DIAS, we have obtained the first dynamic 3-dimensional description of human polymorphonuclear leukocytes (PMN) translocating on a glass surface. A general behavior cycle has emerged which includes two phases. In the first, an ellipsoidal PMN with significant z-axis extends anteriorly and descends to the substratum. When the ventral surface of the anterior end contacts the substratum, there is rapid anterior expansion, which correlates with velocity peaks. In the second phase, the elongate PMN stops translocating along the substratum, the anterior end lifts off of the substratum, sometimes to heights greater than the length of the PMN at the substratum, and finally the PMN retracts into an ellipsoidal morphology still capable of random protrusions. During this second phase, which correlates with velocity troughs, turning usually occurs. The degree of turning is restricted by the continuous integrity of the posterior uropod. The period of the behavior cycle varies from roughly 0.5 to 2 min between PMNs, but is relatively constant within each individual PMN.


Classification of temporomandibular joint osteoarthrosis and internal derangement. 2. Specific diagnostic criteria. Separate entities of temporomandibular joint osteoarthrosis and internal derangement are operationally defined. Criteria are based on an analysis of the diagnostic significance of symptoms and signs, principles of synovial joint pathology in general, and on therapeutic considerations. The primary goal was to enable identification of characteristic stages of osteoarthrosis and internal derangement using diagnostic tools readily available in practice.


Temporomandibular-related pressure thresholds: a model for establishing baselines. The use of pressure threshold measurement to quantify tender spots and trigger points in muscles is well established. Its application to the field of temporomandibular disorders, however, has been more recent and less widely published. Pressure threshold measurement may be useful to define values for dysfunctional muscles or structures so that changes in muscle condition through a course of treatment can be quantified. Normal values for many muscles in the body have been established, but studies of this type in the masticatory system are limited and have often used a fixed, pre-determined pressure cutoff, rather than measuring thresholds across a number of patients. This pilot study seeks to determine whether normal values exist for the anterior temporalis muscles, the masseter muscles, and the lateral capsules of the temporomandibular joints. Intrarater and interrater reliability in measuring thresholds was also studied with the aim of contributing toward a methodological model for further study.


Tinnitus of TMJ origin: a preliminary report. Twenty patients whose chief complaint was tinnitus were examined. They were not known to have temporomandibular disorders. They did not have pain or dysfunction. They were examined by physicians for ear disorders and the results were considered negative. Each of these patients had a complete history and clinical temporomandibular joint examination. The clinical examination included muscle and joint palpation and stethoscopic examination of the joint. This examination also included selected computerized mandibular scans and electromyographic studies of selected facial muscles. Each subject had eight views of transcranial lateral oblique x-rays taken. It was determined that 19 of these individuals had one or more clinical, electromyographic, and radiographic indications of a temporomandibular disorder. From this study, it appears that individuals who have tinnitus with no apparent otologic basis for this symptom should have a careful evaluation of the temporomandibular apparatus. A temporomandibular disorder may be one of the primary causes of this symptom.


Physical therapy post-arthroscopic TMJ management (update). Arthroscopic surgery of the temporomandibular joint is now being widely used for therapeutic, rather than diagnostic, benefits. However, there still remains a void in the literature as to the various rehabilitative protocols used on those patients who have undergone arthroscopy. This article presents a revision of techniques and procedures in the treatment and postoperative management of arthroscopic cases. This revision has been written after five years of experience and the treatment of approximately 300 cases.


Physical therapy evaluation of the craniomandibular pain and dysfunction patient. This article offers an evaluation form to evaluate the craniomandibular pain and dysfunction patient. This form combines dental and physical therapy evaluation components in a two-page format that is quick and easy to use. By using this form physical therapists are able to determine the problem list, develop the treatment plan, and establish excellent communication between the patient, therapist, and dentist.


Contribution of oral habits to dental disorders. Oral habits or parafunction may contribute to dental, periodontal, or neuromuscular damage. Such habits, of which the patient is often unaware, may cause considerable damage. Habits may be occlusal or non-occlusal, and may affect the dentition and/or the oral soft tissues. Drawing a patient's attention to the damage caused by some habits of which he or she is unaware often leads to cessation, whereas with certain conscious habits, such as nail or finger biting, success is much more limited.


A comparative study of three therapeutic modalities in a temporomandibular disorder (TMD) population. A comparative study of three treatment modalities, pharmacologic, occlusal appliance, and their combined use, was conducted to test their therapeutic efficacy on 61 temporomandibular disorder (TMD) patients. Alprazolam (Xanax) was used for the pharmacologic treatment; the occlusal appliance therapy consisted of a flat maxillary stabilization splint. Of the 61 patients, 19 received Alprazolam, 30 received occlusal appliance therapy, and 12 received combined therapy. Subjects were examined at two-week intervals for two months. Only 42 patients attended all follow-up visits. Eight parameters were studied: severity of pain, periodicity of pain, self-evaluated stress, muscle sensitivity to palpation, joint sensitivity to palpation, joint noises, limitation of opening, and limitation of lateral movement. No significant difference was found between the treatment modalities for most of the parameters. All three proved to be effective. Alprazolam increased the restricted mandibular movement, was least effective on joint sensitivity to palpation, and had no effect on joint noises. The combined treatment approach not only failed to prove superior to the other treatments, but showed less improvement in some parameters, possibly due to the small sample.


Organization and patient flow in an urban musculoskeletal dysfunction clinic. A comprehensive clinic for treatment of a variety of musculoskeletal disorders is examined from several points of view. This clinic, affiliated with a comprehensive health sciences university and located in a tertiary medical center, affords efficient access to a variety of health care specialties and subspecialties. Patient sources, severity of dysfunction, and typical patient flow are analyzed in order to describe the clinic's functions.


Multivariate analysis of craniomandibular disorders. This article presents the clinical application of a special statistical method, the Multiple Correspondence Analysis (MCA), derived from data analysis and particularly suited to study a restricted population (here, 202 patients with temporomandibular joint [TMJ] disorders) whose characteristics are defined by a large number of modalities (140). Processing of a Burt-type data table, or Complete Disjunctive Table (CDT), provided a geometrical visualization of information, and permitted determination of the prevalent factor axes. Results showed a significant relationship between all the articular disorders, either peripheral or focal (TMJ), thus revealing the possibility of a yet, little known morphogenic influence on the genesis of TMJ disorders.


Classification of temporomandibular joint osteoarthrosis and internal derangement. 1. Diagnostic significance of clinical and radiographic symptoms and signs. In the clinical setting, diagnosis primarily depends on the history and present status of the patient, and on the examiner's expertise in physical examination and interpretation of conventional radiographs. In order to evaluate the diagnostic significance of clinical and radiographic variables, the authors used magnetic resonance imaging as the 'golden standard' for articular disk position. A total of 90 temporomandibular joints (TMJs) were available for the study. Group I (representing 'normal' disk position) comprised eight joints (8.9%); group II (representing reducing disk), 34 joints (37.8%); and group III (non-reducing disk), 48 joints (53.3%). Nine anamnestic, 15 clinical, and three radiographic variables obeyed pre-defined univariate selection criteria. Relatively high sensitivities were found for clicking-related variables in MR-group II, and for variables related to movement restriction in MR-group III. None of the symptoms or signs appeared to be pathognomonic for either one of the diagnostic groups. Principal component analysis revealed 13 factors that could be grouped into three major categories, representing impairment of joint mechanics, joint pain and tenderness, and radiographically detectable degenerative changes, respectively. Discriminant analyses showed that symptom combinations, which included clinical variables related to joint mechanics, appeared to provide the most useful diagnostic information. It is concluded that reducing and permanent disk displacement can be distinguished in many cases using clinical and radiographic variables. However, there is considerable variability within these groups. To establish a specific clinical diagnosis, a more detailed classification of osteoarthrosis and internal derangement is desirable.


Head posture and cervicovertebral and craniofacial morphology in patients with craniomandibular dysfunction. A relationship between particular characteristics of dental occlusion and craniomandibular disorders (CMD) has been reported, while less attention has been focused on the possible effect of dysfunction of the masticatory system on head posture or cervicovertebral and craniofacial morphology. Natural head position roentgen-cephalograms of 16 young adults with complete dentition taken before and after stomatognathic treatment displayed an extended head posture, smaller size of the uppermost cervical vertebrae, decreased posterior to anterior face height ratio, and a flattened cranial base as compared with age- and sex-matched healthy controls. The lordosis of the cervical spine straightened after stomatognathic treatment. The results are an indication of the close interrelationship between the masticatory muscle system and the muscles supporting the head, and lead to speculation on the principles of treating craniomandibular disorders.


Superficial vascularity of temporomandibular joint retrodiskal tissue: an element of the internal derangement process. Superficial vascular changes in the retrodiskal tissue are an aspect of the remodeling process during progressive anterior displacement of the temporomandibular joint (TMJ) disk. The salient portion of the remodeled retrodiskal tissue (RRT) is contained in a 3 x 3 grid of arthroscopic fields in the superior joint space, plus the tympanic portion of the remodeled retrodiskal tissue. A four-level vascularity classification system gives a statistically significant association between superficial avascularity of the remodeled retrodiskal tissue and progressive stages of disk derangement. In particular, simultaneous avascularity in both the tympanic remodeled retrodiskal tissue and the central arthroscopic field occurs with severe disk displacement without reduction. Analyses are based on 16 microscopic studies, 25 magnetic resonance imaging and tomographic imaging studies, and 26 arthroscopic studies.


Development of alloplastic materials for temporomandibular joint prosthesis: a historical perspective with clinical illustrations. This paper gives the history of the development of alloplastic materials presently used in the temporomandibular joint (TMJ). It discusses the evaluation of TMJ implants, from those being used primarily for ankylosis to the more varied and sophisticated implant designs used today. There is a discussion on the treatment of osteoarthritis with implants and also a discussion on the use of TMJ implants to repair skeletal and dental malrelationships, primarily anterior open bites. Included is a presentation of the latest research by Schellhas et al., which establishes that previously undiagnosed organic disease in the joint proper is a common cause of skeletal and dental malrelationships and that TMJ disease can cause malocclusion.


Surgical treatment of temporomandibular joints in patients with chronic arthritic disease: preoperative findings and one-year follow-up. Twenty-nine temporomandibular joints (TMJs) in 19 patients with chronic arthritic disease were surgically treated. Nine patients had rheumatoid arthritis (including two with juvenile type), six had ankylosing spondylitis and four had psoriatic arthropathy. Using a preauricular approach, diskectomies with synovectomies were performed in 23 joints (14 patients) with chronic arthritic abnormalities. Diskectomies without synovectomies were performed in six joints (five patients), which proved to have internal derangements unrelated to their chronic arthritic disease. Joints with chronic arthritis showed considerable variation in inflammatory reactions, but were characterized by increased vascularization, synovial proliferation to the articulating surfaces and mostly pannus formation and bone resorption. A response in pain relief was seen in 85% of the patients three months postoperatively and in 79% of the patients 12 months postoperatively, indicating that diskectomy with synovectomy may be favorable in patients with severe TMJ problems due to involvement of chronic arthritic disease.


Postoperative physical therapy in temporomandibular joint arthroplasty. This article offers a revised physical therapy treatment approach for rehabilitating a temporomandibular joint (TMJ) following arthroplastic surgical intervention. Surgical repair is only advised in those chronic cases where refractory symptoms affect the quality of the patient's life and prevent patients from carrying out their normal daily activities. Previous nonsurgical and surgical management usually fails to correct the problem in these types of cases. Rehabilitation guidelines discussed include plication, diskectomy (with or without interpositional implant), costochondral graft, fossa implant, and total joint replacement.


Temporomandibular joint osteoarthrosis and generalized joint hypermobility. Peripheral joint mobility of temporomandibular joint (TMJ) osteoarthrosis and internal derangement patients (n = 25) and of a control group (n = 29) was measured according to a rigidly standardized protocol, in order to study the relationships between TMJ osteoarthrosis and internal derangement and generalized joint hypermobility. No significant differences in peripheral joint mobility between the two groups were found. Therefore, generalized joint hypermobility cannot be considered as a predisposing factor for TMJ osteoarthrosis.


A simple questionnaire and clinical examination to help identify possible non-craniomandibular disorders that may influence a patient's CMD symptoms. A patient with craniomandibular disorder (CMD) symptoms may have CMD, a local or systemic disorder that mimics CMD, or CMD superimposed with a local or systemic disorder that exacerbates the CMD symptoms. When evaluating a patient with CMD symptoms, the practitioner needs to determine whether local or systemic problems contribute to the patient's symptoms. The author presents a simple questionnaire that can help identify some non-CMD problems that may contribute to the patient's symptoms. A discussion of each question and examples of possible non-CMD disorders are provided. The author also describes a clinical examination that may be used in conjunction with the questionnaire to help identify some non-CMD conditions.


Conservative and successful treatment of temporomandibular dysfunction in a private rural practice. This clinical study of 382 patients with temporomandibular dysfunction (TMD) details how successful treatment can be achieved in a rural area without the support and services of specialized TMD clinics. Successful treatment is within the scope of the general dentist, provided the practitioner understands the patient's total problem and treatment options. The study spanned a 10-year period, including follow-up at one, five, and eight-year intervals.


Musicians and temporomandibular disorders: prevalence and occupational etiologic considerations. Temporomandibular dysfunction (TMD) appears to be a significant problem affecting musicians. While the incidence of TMD symptoms for musicians is similar to that of the general population, these symptoms are activated and accentuated when performing or practicing. TMD symptoms appear to be significant in trombone, trumpet, tuba, violin, and viola musicians. It is the author's opinion that vocalists are also significantly affected by TMD. Preliminary results of a survey in progress relative to TMD and the singing voice are reported.


Case report of trigeminal neuralgia presenting as odontalgia. A case of trigeminal neuralgia presenting as odontalgia in a 47-year-old female is described. This case shows the difficulty of making a correct diagnosis and how patients can be subjected to unnecessary treatment and continued suffering when a correct diagnosis is not made.


Facial skeleton remodeling due to temporomandibular joint degeneration: an imaging study of 100 patients. One hundred patients with recently acquired, externally visible mandibular deformity and no history of previous extraarticular mandible fracture were selected for retrospective analysis. All had been investigated clinically and with radiography, tomography, and high-field surface-coil MR imaging to determine the presence or absence and extent of temporomandibular joint degeneration. Temporomandibular joint degeneration was found in either one or both joints of each patient studied. Chin deviation was always toward the smaller mandibular condyle or more diseased joint, and many patients either complained of or exhibited malocclusion, often manifested by unstable or fluctuating occlusion disturbances. Three radiologically distinct forms of degenerative vs adaptive osteocartilaginous processes--(1) osteoarthritis, (2) avascular necrosis, and (3) regressive remodeling--involving the mandibular condyle and temporal bone were identified in joints most often exhibiting meniscus derangement. Osteoarthritis and avascular necrosis of the mandibular condyle and temporal bone were generally associated with pain, mechanical joint symptoms, and occlusion disturbances. Regressive remodeling was less frequently associated with occlusion disturbances, despite remodeling of the facial skeleton, and appears to result from regional osteoporosis. Forty patients (52 joints) underwent open arthroplasty procedures, including either meniscectomy or microsurgical meniscus repair, at which time major radiologic diagnoses were confirmed. Surgical and pathologic findings included meniscus displacement, disk degeneration, synovitis, joint effusion, articular cartilage erosion, cartilage healing/fibrosis, cartilage hypertrophy, osseous sclerosis, osteophyte formation, osteochondritis dissecans, localized or extensive avascular necrosis, and decreased mandibular condyle mass and vertical dimension. We conclude that temporomandibular joint degeneration is the principal cause of both acquired facial skeleton remodeling and unstable occlusion in patients with intact dentition and without previous mandible fracture.


A commentary on methods in peptide neurobiology: focus on neurotensin. Research into the identification and functional role of neuropeptides has taken a prominent place in the field of neurobiology within the past 15 years. The type of knowledge available concerning these compounds is, by necessity, limited by the experimental methods currently in use. In this manuscript, an overview is provided of several types of experimental procedures that have been used to study neuropeptide actions in the central nervous system (CNS) and details are presented of the specific application of these procedures as they have been used in the authors' laboratories to study the physiological role of the endogenous brain/gut tridecapeptide neurotensin. The methods were chosen to illustrate research strategies for examining various aspects of peptide neurobiology and include radioimmunoassay (peptide content), high-performance liquid chromatography (HPLC) (analysis of peptide and its fragments), protein phosphorylation (signal transduction), and behavior. Although by no means inclusive, it is the authors' hope that the reader may be able to apply these methods to their own research as well as gain an appreciation for the distance that yet remains on the journey into dissecting the function and physiology of CNS peptides.


Vasopressin involvement in antipyresis, social communication, and social recognition: a synthesis. This review concentrates on the sexually dimorphic neuronal cells and fibers that contain arginine-vasopressin (AVP) and are present in several extrahypothalamic brain areas besides the neurohypophyseal system. Of particular interest are the vasopressinergic neurons that project from the bed nucleus of the stria terminalis and the medial amygdala to the lateral septum because their content of vasopressin is a positive function of circulating levels of testosterone. Physiological and behavioral data suggest that androgen-dependent vasopressin plays an important role in antipyresis and social recognition. In addition, there is evidence that extrahypothalamic AVP-sensitive neurons and hypothalamic AVP-containing neurons control scent marking, a form of social communication in hamsters, in a sex-dependent manner. The interrelationships of these different functions of brain vasopressin are discussed.


The surgical treatment of tendinitis. Clinical rationale and biologic basis. The rationale for surgical treatment of tendinitis is to induce a modulation of the tendon cell-matrix environment by either promoting a renewed wound repair cycle or by the removal of aberrant tissue. The adequacy of healing as a result of surgical tendon treatment has been poorly documented histologically; what evidence exists suggests normal tissue is not achieved. The clinical experience of the authors as well as preliminary analysis of wound healing patterns in a longitudinal internal tenotomy rabbit tendo-Achilles model are discussed.


Overuse injuries to tendons and apophyses in children and adolescents. Overuse injuries to tendons and apophyses are being seen with increasing frequency in children and adolescents as they increase their participation in organized sports, fitness, and dance activities. A retrospective review of 724 overuse tendon and apophyseal injuries seen in the Sports Medicine Clinic at the Boston Children's Hospital suggested that growth and maturation constitute additional risk factors for injury occurrence. Diagnosis and treatment of these injuries is discussed in the context of sports participation.


Complete Achilles tendon ruptures.

Complete Achilles tendon ruptures. Achilles tendon ruptures can be treated nonsurgically in the nonathletic or low-end recreational athletic patient, particularly those more than 50 years of age, provided the treating physician does not delay in the diagnosis and treatment (preferably less than 48 hrs and possibly less than 1 week). The patient should be advised of the higher incidence of re-rupture of the tendon when treated nonsurgically. Surgical treatment is recommended for patients who are young and athletic. This is particularly true because the major criticism of surgical treatment has been the complication rate, which has decreased to a low level and to a mild degree, usually not significantly affecting the repair over time. Surgical treatment in these individuals seems to be superior not only in regard to re-rupture but also in assuring the correct apposition of the tendon ends and in placing the necessary tension on the tendon to secure appropriate orientation of the collagen fibers. This in turn allows them to regain full strength, power, endurance, and an early return to sports. Surgery is also recommended for late diagnosed ruptures where there is significant lengthening of the tendon. Surgical technique should involve a medial incision to avoid the sural nerve, absorbable suture, and augmentation with fascia or tendon where there is a gap or late rupture. Postoperatively, the immobilization should be 7 to 10 days in a splint. A walking boot with early motion in plantar flexion or a short leg cast with the tendon under slight tension should thereafter be used for 4 to 5 weeks. An early and well-supervised rehabilitation program should be initiated to restore the patient to the preinjury activity level.


Partial Achilles tendon tears.

Partial Achilles tendon tears. Persistent pain in the Achilles tendon is often caused by partial ruptures leading to a hypoxic state in the tissues, insufficient healing, and a remaining, immature, and painful scar. The rupture is caused by overuse and conservatively treated with orthotic devices to reduce tendon load and physiotherapy to correct training errors and strength the muscle-tendon unit. Significant partial ruptures seem to respond poorly to conservative measures and do not improve with time. Surgical treatment with excision of degenerated tissue which leads to complete pain relief and full restoration of function with long-standing effects in most cases is why persistent Achilles tendon pain due to partial rupture preferably should be treated surgically.


Achilles tendon overuse injuries.

Achilles tendon overuse injuries. Overuse injuries of the Achilles tendon are common in patients engaging in recreational athletics. Achilles tendon overuse injuries exist as a spectrum of diseases ranging from inflammation of the paratendinous tissue (paratenonitis), to structural degeneration of the tendon (tendinosis), and finally tendon rupture. Factors known to predispose patients to Achilles tendinitis include inadequate stretching, training errors, mechanical malalignment of the lower extremities, rigid training surfaces, and occasionally systemic disease. Treatment of the patient with paratenonitis and tendinosis is initially conservative, emphasizing passive stretching, concentric and eccentric strengthening, correction of training errors, and restoration of normal limb alignment. Patients resistant to this protocol often exhibit a more advanced degree of tendon change. A high percentage of these patients can benefit from surgical debridement of the involved tendon, and they can anticipate successful return to recreational athletics. Steroid injections should not be routinely used in patients with Achilles tendinitis. Rupture of the Achilles tendon following intratendinous injection has been reported.


Injuries at the myotendinous junction.

Injuries at the myotendinous junction. The complete role of the myotendinous junction is discussed in this article. The morphology and function of the junction, typical injuries occurring at this region, delayed-onset muscle soreness, and muscle strain injury are described. Muscle strain injury is covered in detail, including characterization, treatment, and prevention. Clinical information is conveyed as well as basic science study results pertinent to the clinical situation.


Patellar tendon injuries.

Patellar tendon injuries. Patellar tendon injury secondary to overuse represents a failure of soft tissue healing that can lead to chronic symptoms. Clinical staging of this process is helpful in the initial management. Diagnostic imaging can help define the character of the injury as well as localized pathology. Initial treatment of clinical stages I, II, and III is conservative.


Tendon and muscle injuries in the groin area. Groin injuries in athletes are being recognized as one of the most difficult problems in sport. The symptoms associated with chronic groin injuries are often diffuse and uncharacteristic. These injuries usually constitute a major diagnostic and therapeutic challenge, the success of which depends on a correct diagnosis. It is necessary to establish a broad, differential background, and therefore, this article includes a broad description of groin injuries.


Hand tendon injuries in athletics.

Hand tendon injuries in athletics. Hand tendon injuries may be caused by acute trauma or chronic overuse. Although these injuries are common in athletics, specific diagnoses can be difficult to make and are often delayed. Early diagnosis is the key to a good outcome. Most hand tendon injuries do well with nonoperative treatment when initiated early. Long-term disruption of the delicate balance of muscle forces in the hand will lead to secondary changes that are often difficult to correct. Delay in treatment of overuse injuries may produce chronic inflammation that does not respond to conservative care and requires operative management. A sound knowledge of hand anatomy, awareness of the differential diagnosis, and a high index of suspicion are needed for proper care of the athletic hand injury.


Elbow tendinosis/tennis elbow.

Elbow tendinosis/tennis elbow. The histology of pathologic tennis elbow tissue reveals noninflammatory tissue, thus the term angio-fibroblastic tendinosis. The goal of nonsurgical treatment is a revascularization and collagen repair of this pathologic tissue by rehabilitative exercise. In the event of rehabilitation failure, surgical correction by removal of this pathologic tissue is the surgical technique of choice.


Shoulder tendinoses.

Shoulder tendinoses. The most common source of shoulder pain is soft-tissue dysfunction. Although fractures of the humerus, scapula, and clavicle are common throughout life, most problems encountered between the ages of 15 and 60 are related to the ligaments, tendons, and muscles of the shoulder girdle. This article deals mainly with tendinitis and tendinosis as primary entities, and to a lesser extent with tendon problems attributable to a primarily bony or ligamentous dysfunction.


[The status of children and family relations after successful in vitro fertilization and embryo transfer]. Using a retrospective questionnaire method, the authors evaluated the neuropsychologic status of 20 IVF children and the family relationships. The status of the children is favourable and in certain activities (composition of sentences) the children were better than their peers. According to the authors this is due to the increased care of mature parents after the arrival of the long expected child. The authors confirmed on their own material that the birth of an IVF child has a far reaching positive biological, psychological and social impact on both parents and training of their children. If the family environment was influenced by long lasting treatment of sterility, the arrival of an IVF child normalizes the situation.


[Personal experience with colpoureteral suspension using the Burch method]. The objective of the work was to evaluate one approach of evaluation of the effect achieved by the mentioned corrective operation, i.e. the subjective evaluation by the patients themselves. In order to ensure comparability of evaluation at different time intervals, the authors used a questionnaire with 13 questions focused on continence and changes of micturition as well as on sexual aspects and changes in the social sphere. Sixty-nine patients were at least one year after operation. Evaluation 6 weeks after operation was implemented in 105 patients. The subjective sensation of complete recovery and satisfaction with treatment in 93.33% after six weeks and 94.20% after one year with minimal complaints regarding micturition indicates that the operation sec. Burch is a very good corrective operation, resolving stress incontinence of urine in women.


[Evaluation of the analgesic effect of dolsin during labor]. The authors evaluate the analgetic action of 50 mg dolsin in parturient women during childbirth using a Slovak version of McGill's questionnaire on pain. The intensity of pain in women is evaluated before administration of dolsin and after its administration. The obtained data on the analgetic effect are compared with the total score of pain and individual components of pain by the paired t-test. Although the mean values of the total score of pain and the sensory components of pain declined after administration of dolsin, the change was not statistically significant. In the affective component of pain the mean value of PRI(A) increased after dolsin administration.


[Vaginal adenosis and problems in its treatment]. The authors investigated four patients with suspected vaginal adenosis without prenatal exposure to DES. Adenosis was confirmed by histological examination in two instances. In one case the adenosis was formed by glands of the endometrial type. In the second case it was formed by glands of the endocervical type with atypical features. On check-up biopsy an adenocarcinoma was revealed (non-light cell) resembling morphologically a cervical adenocarcinoma. The authors discuss the embryogenesis, histogenesis, epidemiology and differential diagnosis of adenosis. At the same time the authors reflect on problems of its therapy. Finally the authors summarize some practical recommendations for diagnostic and therapeutic purposes.


A single amino-acid change in the iron-sulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis. The sequence of an allele encoding the iron-sulphur protein (Ip) subunit of succinate dehydrogenase (Sdh) was determined following PCR amplification of genomic DNA from a carboxin (Cbx)-sensitive Ustilago maydis strain. Comparison of this sequence with that of the Ip allele from a Cbx-resistant strain (IPr) revealed a two-base difference between the sequences. This mutation led to the substitution of a leucine residue for a histidine residue within the third cysteine-rich cluster of the deduced amino-acid sequence of the Ipr allele. This cluster, which is associated with the S3 iron-redox centre, is involved in the transport of electrons from succinate to ubiquinone (Q). Confirmation that this nucleotide change led to enhanced resistance to Cbx was obtained following mutagenesis of the sensitive Ip allele to the resistant form and expression of the mutated allele in U. maydis.


Organization of ATPA coding and 3' flanking sequences associated with cytoplasmic male sterility in Phaseolus vulgaris L. A region of the mitochondrial genome associated with cytoplasmic male sterility (CMS) in Phaseolus vulgaris was flanked by two different repeated sequences designated x and y. The DNA sequence of the CMS-unique region and a portion of each flanking repeat was determined. Repeat x contained a complete coding copy of the F1 ATPase subunit A (atpA) gene, as well as an open reading frame (orf) predicting a protein of 209 amino acids. The TGA termination codon of the atpA gene and the ATG initiation codon of orf209 were overlapping. These reading frames were oriented with their 3' ends proximal to the CMS-unique region. The CMS-unique region of 3736 nucleotides contained numerous orfs. The longest of these predicted proteins being of 239, 98 and 97 amino acids. The 3' coding and 3' flanking regions of orf98 were derived from an internal region of the higher plant chloroplast tRNA alanine intron. The region of repeat y immediately adjacent to the CMS-unique region contained the 111 carboxy-terminal coding residues of the apocytochrome b (cob) gene. This segment was oriented with its 5' end proximal to the CMS-unique region, but cob gene sequences were not fused to an initiation codon within the unique region.


Involvement of a large inverted repeated sequence in a recombinational rearrangement of the mitochondrial genome of the higher fungus Agrocybe aegerita. Southern hybridization of the total DNA of Agrocybe aegerita with cloned mitochondrial (mt) probes revealed a sequence homology between two distant mitochondrial restriction fragments. From the mtDNA restriction map and the distribution of restriction sites on the cross-hybridizing mitochondrial fragments, two copies of a large inverted repeated sequence (IR) of 3 kbp were located on the mitochondrial genome. These IR sequences divided the 80 kbp mtDNA into two single-copy regions of 24 kbp (SSC) and 50 kbp (LSC). For the first time in higher fungi, this IR sequence has been shown to be involved in an intramolecular homologous recombinational event. Such a rearrangement led to an inversion of the orientation of the two unique-copy regions, without any change in mtDNA complexity. The location of the recombinational event was compared with previously reported plant and fungal mitochondrial rearrangements and the potential role of the IR sequence was discussed.


Cell type determines plastid transmission in tomato intergeneric somatic hybrids. Mesophyll (M)- and suspension culture (S)-derived protoplasts of both Lycopersicon esculentum, tomato, and its wild relative Solanum lycopersicoides were fused as S + M, M + M and S + S combinations, respectively, to resolve the role of parental cell types in determining cpDNA transmission to intergeneric somatic hybrid plants. The mesophyll cpDNA was preferentially transmitted to 96% of the plants, each regenerated from a separate callus, in M + S and S + M fusion combinations. In contrast, for the M + M combination there was an equable distribution of either tomato cpDNA or that of S. lycopersicoides among the 34 hybrid plants. The number of plastids or proplastids in mesophyll or suspension protoplasts was not a factor regulating cpDNA transmission. Mesophyll or suspension protoplasts of both fusion partners had comparable frequencies of either plastid type with a mean of 23. The biased transmission of plastids from the mesophyll parent in somatic hybrid plants of S + M and M + S combinations appears to be due to differential multiplication of plastids, possibly conditioned by an unequal input of the nucleoids found in plastids versus proplastids. In the M + M fusion, plastid and nucleotide input and subsequent plastid multiplication are apparently equal, and when combined with random sorting out leads to an equal distribution of parental cpDNAs in the regenerated somatic hybrid plants. For the S+S combination, 22 somatic hybrid plants have exclusively tomato cpDNA, an outcome that is not readily explained by donor cell input.


DNA translocations contribute to chromosome length polymorphisms in Candida albicans. Rotating-gel electrophoresis and DNA hybridization were used to compare the electrophoretic karyotype of six Candida albicans isolates. The hybridization pattern for 22 cloned sequences, including eight previously unmapped genes, indicates that there are eight pair of homologous chromosomes in each strain. However, since homologous chromosomes can differ in length, it is possible to resolve more than eight bands in some strains. The mapping data demonstrate that linkage groups are generally conserved suggesting that, in spite of gross karyotype differences, there is an underlying similarity in the genome organization of different isolates. The hybridization data also provide direct evidence that DNA translocations and reciprocal translocations contribute to chromosome length polymorphisms in C. albicans.


Complementation of Saccharomyces cerevisiae acid phosphatase mutation by a genomic sequence from the yeast Yarrowia lipolytica identifies a new phosphatase. A Yarrowia lipolytica gene library was constructed in vector YRp7 and transformed into a Saccharomyces cerevisiae strain lacking both major acid phosphatase activities. A 2.18 kb genomic sequence restoring the ability to hydrolyze alpha-naphthyl phosphate was isolated. Its sequencing revealed an ORF encoding 358 amino acids without significant homology with any known phosphatase. A putative signal peptide and several possible sites for N-glycosylation were identified. Phosphate-regulated expression of the cloned gene was observed in Y. lipolytica. Disruption data favoured the hypothesis that it might encode a minor phosphatase species.


Enhanced stability of YEp plasmids in lager brewing yeasts is related to lager brewing yeast 2-microns DNA. YEp plasmid stability in the presence of either Saccharomyces cerevisiae laboratory strain 2-microns DNA, or lager brewing yeast 2-microns DNA in the same genetic background, was compared under non-selective culture conditions. It was found that YEp plasmids were more stably maintained in the presence of lager 2-microns DNA under these conditions. By construction of laboratory-lager 2-microns DNA hybrid plasmids, an 867 bp StuI fragment of lager 2-microns DNA was shown to be responsible for the enhanced stability of the YEp plasmid. Nucleotide substitutions at two sites were found by sequencing this region. It was also confirmed that increasing cell ploidy enhanced YEp stability under non-selective conditions.


Direct induction of tetraploids or homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydrostatic pressure. Hydrostatic pressure and a dye plate method were used to investigate the direct induction of tetraploids or homozygous diploids from the industrial diploid or haploid yeast Saccharomyces cerevisiae. Above 200 MPa, hydrostatic pressure greatly inactivated the strains HF399s1 (alpha haploid), P-540 (a/alpha diploid), and P-544 (a/alpha diploid). At the same time, when pressure-treated cells of these strains were spread on a dye plate, some of the visible colonies were stained red/blue or dark blue (variant colonies); the rest stained violet, similar to colonies originating from diploid cells or haploid cells that were not pressure-treated. In addition, above 100 MPa, the formation of variant colonies increased with increasing pressure, and maximized (1 x 10(-1)) at 200 and 250 MPa, respectively. The size of almost all variant cells from P-544, P-540, and HF399s1 was visibly increased compared with that of untreated cells and the measured cellular DNA content of P-540 and HF399s1 was double that of untreated cells. Furthermore, based on random spore analysis and mass-matings, induced variants in the diploid strains were found to be tetraploid with an a/a/alpha/alpha genotype at the mating-type locus or, in the haploid strains, homozygous diploid with an alpha/alpha genotype. From these results we conclude that pressure treatment in combination with a dye plate is a useful method for strain improvement by direct induction of tetraploids or homozygous diploids from industrial strains whether diploid or haploid.


Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants. The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.


Genetic organization and structural features of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa. The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.


Transformation of Acremonium coenophialum, a protective fungal symbiont of the grass Festuca arundinacea. Acremonium coenophialum is a mutualistic mycosymbiont and natural agent of biological protection of the widely distributed grass Festuca arundinacea (tall fescue). An electroporative transformation system was developed for A. coenophialum. Segments of DNA 5' to the beta-tubulin gene (tub2) of the closely related ascomycete Epichloë typhina, fused to the Escherichia coli hph gene encoding hygromycin B phosphotransferase, conferred hygromycin resistance when introduced into A. coenophialum by electroporation. The incorporation of the Emericella nidulans trpC terminator greatly increased protoplast germination on selective medium and improved transformation efficiencies 30-200% depending on the plasmid construct. Plasmid pCSN43, which incorporates the trpC controlling elements for hph expression, was also used to transform A. coenophialum. Southern blot analysis of ten pCSN43 transformants indicated the possibility of random integration of this vector into the genome.


Genomic organization of a cellulase gene family in Phanerochaete chrysosporium. Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene (cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were found to hybridize to at least three different chromosomes, one of which is dimorphic. The organization of these genes was similar in another P. chrysosporium strain, ME 446. It is clear that, unlike T. reesei, the most well-studied cellulolytic fungus, P. chrysosporium contains a complex, cbh1-like gene family.


Length heterogeneity in ITS 2 and the methylation status of CCGG and GCGC sites in the rRNA genes of the genus Peronosclerospora. The polymerase chain reaction (PCR) was used with primers complementary to conserved flanking sequences to amplify the internal transcribed spacer 2 (ITS 2) of the rDNA repeat units of five Peronoscleropora isolates, one each of P. sorghi, P. maydis, P. sacchari and two of P. zeae. In contrast to the situation found in most-fungi that have been examined, length heterogeneity was evident in each sample. The rDNA composition of the amplified bands was confirmed by Southern hybridizations using an ITS 2 amplified from P. sorghi and cloned rDNA from Neurospora crassa as probes. Length heterogeneity was also detected in genomic DNA digests using the same probes. In addition to one dominant fragment for each isolate, there were several less frequent fragments of different sizes, and the isolate(s) for each species had a unique banding pattern for ITS 2. The absence of 5-methylcytosine residues in CCGG and GCGC sequences in the ribosomal genes of these four Peronosclerospora species was demonstrated by the production of identical banding patterns with ribosomal DNA probes following digestion of genomic DNA with MspI and HpaII, and by complete digestion with CfoI.


Identification of a chloroplast-encoded secA gene homologue in a chromophytic alga: possible role in chloroplast protein translocation. SecA is one of seven Sec proteins that comprise the prokaryotic protein translocation apparatus. A chloroplast-encoded secA gene has been identified from the unicellular chromophytic alga Pavlova lutherii. The gene predicts a protein that is related to the SecA proteins of Escherichia coli and Bacillus subtilis. The presence of secA, as well as the previously described secY and hsp70 genes, on the chloroplast genome of P. lutherii suggests that this eukaryotic organism utilises protein translocation mechanisms similar to those of bacterial cells.


The electrophoretic banding pattern of the chromosomes of Pichia stipitis and Candida shehatae. The electrophoretic karyotype of fu1ur strains of P. stipitis and five strains of C. shehatae were compared by means of OFAGE and TAFE techniques. Although the number of chromosomal bands was six in all cases except one, P. stipitis revealed a clearly distinct pattern in comparison to C. shehatae. Both yeasts showed remarkable chromosome length polymorphism.