Structurally similar Drosophila alpha-tubulins are functionally distinct in vivo

We used transgenic analysis in Drosophila to compare the ability of two structurally similar alpha-tubulin isoforms to support microtubule assembly in vivo. Our data revealed that even closely related alpha-tubulin isoforms have different functional capacities. Thus, in multicellular organisms, even small changes in tubulin structure may have important consequences for regulation of the microtubule cytoskeleton. In spermatogenesis, all microtubule functions in the postmitotic male germ cells are carried out by a single tubulin heterodimer composed of the major Drosophila alpha-84B tubulin isoform and the testis-specific beta 2-tubulin isoform. We tested the ability of the developmentally regulated alpha 85E-tubulin isoform to replace alpha 84B in spermatogenesis. Even though it is 98% similar in sequence, alpha 85E is not functionally equivalent to alpha 84B. alpha 85E can support some functional microtubules in the male germ cells, but alpha 85E causes dominant male sterility if it makes up more than one-half of the total alpha-tubulin pool in the spermatids. alpha 85E does not disrupt meiotic spindle or cytoplasmic microtubules but causes defects in morphogenesis of the two classes of singlet microtubules in the sperm tail axoneme, the central pair and the accessory microtubules. Axonemal defects caused by alpha 85E are precisely reciprocal to dominant defects in doublet microtubules we observed in a previous study of ectopic germ-line expression of the developmentally regulated beta 3-tubulin isoform. These data demonstrate that the doublet and singlet axoneme microtubules have different requirements for alpha- and beta-tubulin structure. In their normal sites of expression, alpha 85E and beta 3 are coexpressed during differentiation of several somatic cell types, suggesting that alpha 85E and beta 3 might form a specialized heterodimer. Our tests of different alpha-beta pairs in spermatogenesis did not support this model. We conclude that if alpha 85E and beta 3 have specialized properties required for their normal functions, they act independently to modulate the properties of microtubules into which they are incorporated.     

Imidazoline receptor proteins are regulated in platelet-precursor MEG-01 cells by agonists and antagonists

A morphological analysis was done of 15 cases of malignant cerebral lymphomas selected from the material of 160 brains of patients, who died in the course of full-blown acquired immune deficiency syndrome (AIDS) during the period of 1987-1997. Cases with cerebral lymphomas comprised 9.4% of the whole collection. There were 13 males and 2 females in the studied group. The patients age ranged from 25 to 61 years. In 10 cases lymphomas were localized solely in the central nervous system, and in further 4 they were accompanying systemic neoplastic process. In one case lack of clinical and autopsy data did not permit classification of neoplasm to the primary or to the secondary group. In 13 cases immunophenotype of the lymphomas was characterized by immunohistochemical methods. In 11 cases neoplastic cells originated from B cells line and in 2--from T cells line. In 10 cases lymphomas were found in macroscopic examination, in the remaining 5 cases they were disclosed at the brain histopathology. The dynamics and extensiveness of the neoplastic process were different in particular cases. In most of them the process was multifocal and manifested in the form of diffuse proliferation, formed tumors with changing nature of their delineation and as multilayer perivascular cuffs. The characteristic feature of diffuse neoplasmatic growth was the appearance of large coagulative necroses in the central parts of tumors. Neoplastic foci were localized most often in the cerebral hemispheres (white matter, basal ganglia, periventricular regions), less frequently in the brain stem and cerebellum. In one case diffuse lymphoid growth involved selectively leptomeninges. In most of the cases leptomeningeal infiltrations accompanied large parenchymal neoplastic foci. The most striking feature of our collection consisted in concomitance of cerebral lymphomas with HIV-specific brain pathology and/or opportunistic infections mostly of viral etiology. Their frequency was much higher than in cases of AIDS without cerebral lymphomas. Another finding which seems to be worth mentioning was the appearance of morphological exponents of various pathological processes such as for instance multinuclear giant cells, CMV inclusions within neoplastic tissue. The relatively frequent presence of numerous HIV-specific giant cells on the periphery of lymphomatous tumors suggests pathogenetic participation of immune deficiency virus in the blastomatous transformation of lymphoid cells within the central nervous system.     

Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae

Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer. In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homologous) sequences. We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homologous sequences. We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence. Defects in mismatch repair can elevate homologous recombination between 91% homologous substrates as much as 100-fold while having only modest effects on recombination between 77% homologous substrates. These observations have implications for genome stability and general mechanisms of recombination in eukaryotes.     

A postsynaptic excitatory amino acid transporter with chloride conductance functionally regulated by neuronal activity in cerebellar Purkinje cells

Excitatory amino acid (EAA) neurotransmitters induce postsynaptic depolarization by activating receptor-mediated cation conductances, a process known to underlie changes in synaptic efficacy. Using a patch-clamp method, we demonstrate here an EAA-dependent postsynaptic anion conductance mediated by EAA transporters present on cerebellar Purkinje cell bodies and dendrites in culture. This transporter-mediated current was modulated by neuronal activity: it exhibited facilitation for >20 min after transient depolarization accompanied by Ca2+ influx. Evidence is presented suggesting that the transporter facilitation is mediated by arachidonate release after Ca2+-dependent activation of phospholipase A2, which exists in Purkinje cells. This postsynaptic reuptake system may represent a novel modulatory mechanism of synaptic transmission as well as prevent neuronal excitotoxicity.     

Modulation of cell surface fibronectin assembly sites by lysophosphatidic acid

Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet-poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino-terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing.     

Identification of proteins that are abnormally regulated in differentiated cultured human keratinocytes

The inhabitants living in the neighbourhood of a deserted mercury-contaminated industrial site are subjected to an age-group differentiated mercury exposure assessment based on a scenario-linked calculation. Analytical input data for the calculation procedure are provided for from soil, air and plants in a large number. The most sensitive group are small children being mainly exposed by soil ingestion which makes up nearly 80% of the ADI, followed by inhalation of mercury contaminated indoor air. On the other hand, inhalation of indoor air has a predominant impact on youth and adults.     

Identification and characterization of a lysophosphatidic acid receptor

A specific binding site for 1-[3H]stearoyl-lysophosphatidic acid (stearoyl-LPA) was identified and characterized in membranes prepared from rat brain and Swiss 3T3 fibroblasts. Specific binding of [3H]LPA to these sites was protein dependent, was saturable, reached equilibrium in 15 min, and was displacable by the addition of excess unlabeled LPA. Scatchard analysis of saturation binding experiments indicated that these sites had affinities of 2.0 +/- 0.5 nM and 5.4 +/- 2.6 nM and densities of 19 +/- 3 fmol/micrograms of protein and 38 +/- 6 fmol/micrograms of protein in rat brain and 3T3 cell membranes, respectively. Various LPAs, with different acyl groups in the sn-1-position, competed with [3H]LPA for these binding sites, with a rank order of potency of 1-oleoyl-LPA > 1-stearoyl-LPA = 1-palmitoyl-LPA > 1-myristoyl-LPA. Phosphatidic acid also bound to these sites, but with lower affinity than any LPA tested. Neither lysophosphatidylcholine, lysophosphatidylethanolamine, nor any free fatty acid competed with [3H]LPA for these binding sites. Binding of [3H]LPA to these sites was regulated by nonhydrolyzable guanine nucleotides in both rat brain and 3T3 cell membranes. Furthermore, in 3T3 cells, these sites were regulated by cell density. It was subsequently determined that LPA induced a transient increase in intracellular Ca2+ levels in 3T3 cells. The concentrations required for this response, as well as the rank order of potency of the various LPAs and phosphatidic acid, correlated with the affinity of these compounds for the [3H]LPA binding site. These results suggest that the specific, high affinity, binding sites for [3H]LPA are G protein-coupled receptors.     

Two-dimensional gel electrophoresis of actin-binding proteins isolated by affinity chromatography from human skeletal muscle

Obstructive azoospermia represents approximately 10 % of cases of male hypofertility. It is classified according to the volume of ejaculate. When the latter is normal a proximal obstruction is suspected. Scrotal sonography can help to detect dilation of the epididymal head when clinical findings are equivocal. Ejaculatory duct obstruction (EDO) is suspected when the volume of ejaculate is low. The use of transrectal ultrasonography (TRUS) plays a major role in the investigation of these patients, and endorectal MRI is a very useful adjunct in selected cases. The most common cause of EDO is congenital bilateral absence of vas deferens, which is now thought to be a genital form of cystic fibrosis in 80 % of cases. Consequently, a definitive diagnosis must be made before any attempt at in vitro fertilization. TRUS accurately visualizes abnormalities of the caudal junction of the vas deferens and seminal vesicles, yielding a definitive diagnosis without scrototomy. Other causes of EDO are congenital cysts compressing the distal part of the ejaculatory ducts and inflammatory distal stenosis. The former are accurately identified by TRUS, but the latter give more or less marked signs of obstruction which are only of value in azoospermic patients with a low-volume ejaculate. More invasive imaging is required to diagnose partial obstruction of the ED. Surgical vasography is still the reference, but puncture of the seminal vesicles under TRUS guidance is an attractive alternative, as it permits aspiration of seminal fluid (to seek motile sperm) and vasography without scrototomy. Lastly, endorectal MRI well assesses the relationships between the proximal prostatic urethra and the posterior wall of the ejaculatory ducts, which need to be precisely known when endoscopic resection of the ejaculatory ducts is planned.     

How to analyze electron micrographs of rafts of actin filaments crosslinked by actin-binding proteins

Actin bundles are common cytoskeletal structures but ones which are usually polymorphic, varying from bundle to bundle. Two-dimensional arrays of actin filaments crosslinked by actin-bundling proteins are more tractable structures to analyze than are the three-dimensional bundles found in cells. The first step in analyzing these two-dimensional "rafts" is to determine the spatial relationships between neighboring filaments. It is difficult to discern such relationships by inspection of the electron micrographs of rafts, but easy by examination of the Fourier transforms. We provide theory and examples of the analysis of transforms of rafts, and show that different bundling proteins give rise to different kinds of rafts.     

Chimeric rRNAs containing the GTPase centers of the developmentally regulated ribosomal rRNAs of Plasmodium falciparum are functionally distinct

The human malaria parasite, Plasmodium falciparum, maintains at least two distinct types, A and S, of developmentally controlled ribosomal RNAs. To investigate specific functions associated with these rRNAs, we replaced the Saccharomyces cerevisiae GTPase domain of the 25S rRNA with GTPase domains corresponding to the Plasmodium A- and S-type 28S rRNAs. The A-type rRNA differs in a single nonconserved base pair from the yeast GTPase domain. The S-type rRNA GTPase domain has three additional changes in highly conserved residues, making it unique among all known rRNA sequences. The expression of either A- or S-type chimeric rRNA in yeast increased translational accuracy. Yeast containing only A-type chimeric rRNA and no wild-type yeast rRNA grew at the wild-type level. In contrast, S-type chimeric rRNA severely inhibited growth in the presence of wild-type yeast rRNA, and caused lethality in the absence of the wild-type yeast rRNA. We show what before could only be hypothesized, that the changes in the GTPase center of ribosomes present during different developmental stages of Plasmodium species can result in fundamental changes in the biology of the organism.     

Laminin-like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro

OBJECTIVE: To review data supporting the hypothesis that syndrome X plays a major role in the pathogenesis of coronary artery disease (CAD), and the effects of lifestyle factors and pharmacologic interventions on insulin, other metabolic parameters, and outcomes. DATA SOURCES: MEDLINE (January 1966-August 1997) and Current Contents database searches identified applicable English-language experimental trials, epidemiologic studies, reviews, and editorials. STUDY SELECTION AND DATA EXTRACTION: Studies that were included addressed the role of insulin resistance and hyperinsulinemia in the pathogenesis of CAD or the effects of lifestyle factors and pharmacologic interventions on metabolic parameters and outcomes. DATA SYNTHESIS: The main characteristics of syndrome X are hyperinsulinemia and insulin resistance. These result in secondary syndrome X features, including hyperglycemia, increased very-low-density lipoprotein concentrations, decreased high-density lipoprotein cholesterol, and hypertension. Insulin resistance is worsened by obesity, and insulin has been shown to contribute to the development of hypertension. Other studies demonstrate that smoking adversely affects glucose and insulin concentrations. Animal studies have linked hyperinsulinemia and atherogenesis. These animal data have been confirmed by several large prospective and population studies that have identified associations between hyperinsulinemia and CAD. CONCLUSIONS: Strong evidence links insulin resistance and hyperinsulinemia to CAD. Lifestyle modifications play an important role in decreasing cardiovascular risk, and clinicians should strongly encourage such changes. Clinicians must also carefully consider the effects of antihypertensive, antihyperglycemic, and antidyslipidemic agents on patients' metabolic profiles when choosing appropriate therapeutic regimens. However, outcome data on many potentially beneficial agents, including calcium antagonists, alpha 1-adrenergic antagonists, angiotensin-converting enzyme inhibitors, metformin, acarbose, and troglitazone, are not yet available.     

Stress-activated protein kinases are negatively regulated by cell density

Stimulation by UV irradiation, TNFalpha, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts. This results in the phosphorylation of the N-terminal domain of c-Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c-Jun phosphorylated at Ser63, we show that culture confluency drastically inhibited c-Jun N-terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G-proteins cdc42 and Rac1. In contrast, the classical MAPK pathway was insensitive to confluency. The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c-Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress-dependent signal cascade by confluency will restrict c-Jun N-terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area.     

Functional analyses of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally

The biphenyl dioxygenases (BP Dox) of strains Pseudomonas pseudoalcaligenes KF707 and Pseudomonas cepacia LB400 exhibit a distinct difference in substrate ranges of polychlorinated biphenyls (PCB) despite nearly identical amino acid sequences. The range of congeners oxidized by LB400 BP Dox is much wider than that oxidized by KF707 BP Dox. The PCB degradation abilities of these BP Dox were highly dependent on the recognition of the chlorinated rings and the sites of oxygen activation. The KF707 BP Dox recognized primarily the 4'-chlorinated ring (97%) of 2,5,4'-trichlorobiphenyl and introduced molecular oxygen at the 2',3' position. The LB400 BP Dox recognized primarily the 2,5-dichlorinated ring (95%) of the same compound and introduced O2 at the 3,4 position. It was confirmed that the BphA1 subunit (iron-sulfur protein of terminal dioxygenase encoded by bphA1) plays a crucial role in determining the substrate selectivity. We constructed a variety of chimeric bphA1 genes by exchanging four common restriction fragments between the KF707 bphA1 and the LB400 bphA1. Observation of Escherichia coli cells expressing various chimeric BP Dox revealed that a relatively small number of amino acids in the carboxy-terminal half (among 20 different amino acids in total) are involved in the recognition of the chlorinated ring and the sites of dioxygenation and thereby are responsible for the degradation of PCB. The site-directed mutagenesis of Thr-376 (KF707) to Asn-376 (LB400) in KF707 BP Dox resulted in the expansion of the range of biodegradable PCB congeners.     

Dendritic cell survival and maturation are regulated by different signaling pathways

OBJECTIVE: Quantification of serum nucleotide pyrophosphohydrolase (NTPPHase) activity in healthy subjects and in patients with various rheumatic diseases or with quad/hemiplegia, hemodialysis, or renal transplant. METHODS: Colorimetric assay of enzyme activity in serum. RESULTS: Serum NTPPHase activity in 85 healthy subjects was independent of age or sex and was highly reproducible in each individual. The biologic and methodologic coefficients of variation were nearly identical. Elevated enzyme levels were found in sera from patients with osteoarthritis/spondylosis, calcium pyrophosphate dihydrate (CPPD) crystal deposition, scleroderma, fibromyalgia, or hemodialysis. Renal transplant patients receiving cyclosporine had the highest enzyme activity of any group, whereas transplant patients not taking this drug had normal levels. Histograms of values in all groups showed a normal distribution. CONCLUSION: Serum NTPPHase activity levels were significantly elevated in patients with degenerative arthritis whether or not CPPD crystals were present, in patients with either scleroderma or fibromyalgia, and in patients receiving hemodialysis therapy or taking cyclosporine.     

Cytosolic and nuclear mitogen-activated protein kinases are regulated by distinct mechanisms

We have investigated the regulation and localization of mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) in both cytosolic and nuclear fractions of glomerular mesangial cells. p42 MAPK was localized by both immunoblot and kinase activity in both cytosol and nucleus and was rapidly activated, in both fractions, by fetal bovine serum and TPA. Downregulation of protein kinase C (PKC) by TPA inhibited stimulation of cytosolic p42 MAPK, but unexpectedly had no effect on stimulated p42 MAPK in the nucleus. Next we studied the upstream kinase p45 MAPKK by indirect immunofluorescence microscopy, Western blot analysis, and kinase specific activity. Unlike MAPK, p45 MAPKK is almost exclusively cytosolic in resting cells and kinase activity stimulated by TPA is restricted to the cytosol. Interestingly, PKC downregulation for 24 h with TPA dramatically enhanced nuclear MAPKK as assessed by all three techniques. Cytosolic stimulated MAPKK was attenuated in PKC downregulation. Collectively these results show that in mesangial cells: (i) p42 MAPK and p45 MAPKK localize in both the cytosol and the nucleus, and (ii) PKC exerts a negative effect on nuclear MAPKK activity as documented by PKC downregulation, which augments p45 MAPPK nuclear mass and activity. These results indicate that the dual regulation of these two kinases is under differential control in the cytosol and the nucleus.     

Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation

The capacity of dendritic cells (DC) to initiate immune responses is dependent on their specialized migratory and tissue homing properties. Chemotaxis and transendothelial migration (TEM) of DC were studied in vitro. Immature DC were generated by culture of human monocytes in granulocyte-macrophage colony-stimulating factor and IL-4. These cells exhibited potent chemotaxis and TEM responses to the CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and monocyte chemotactic protein-3, and weak responses to the CC chemokine MIP-3beta and the CXC chemokine stromal cell-derived factor (SDF)-1alpha. Maturation of DC induced by culture in lipopolysaccharide, TNF-alpha or IL-1beta reduced or abolished responses to the former CC chemokines but markedly enhanced responses to MIP-3beta and SDF-1alpha. This correlated with changes in chemokine receptor expression: CCR5 expression was reduced while CXCR4 expression was enhanced. These findings suggest two stages for regulation of DC migration in which one set of chemokines may regulate recruitment into or within tissues, and another egress from the tissues.     

Three proteins involved in Caenorhabditis elegans vulval invagination are similar to components of a glycosylation pathway

We have molecularly analyzed three genes, sqv-3, sqv-7, and sqv-8, that are required for wild-type vulval invagination in Caenorhabditis elegans. The predicted SQV-8 protein is similar in sequence to two mammalian beta(1,3)-glucuronyltransferases, one of which adds glucuronic acid to protein-linked galactose-beta(1, 4)-N-acetylglucosamine. SQV-3 is similar to a family of glycosyltransferases that includes vertebrate beta(1, 4)-galactosyltransferases, which create galactose-beta(1, 4)-N-acetylglucosamine linkages. One model is therefore that SQV-8 uses a SQV-3 product as a substrate. SQV-7 is similar to members of a family of nucleotide-sugar transporters. The sqv genes therefore are likely to encode components of a conserved glycosylation pathway that assembles a C. elegans carbohydrate moiety, the absence of which perturbs vulval invagination.     

Rho guanosine triphosphatase mediates the selective stabilization of microtubules induced by lysophosphatidic acid

We have examined the ryanodine receptor, Ca(2+)-ATPase, calsequestrin and phospholamban mRNA levels in the left ventricles of pacing-induced heart failure and norepinephrine infusion dogs. The heart failure dogs showed a decrease in the levels of ryanodine receptor and Ca(2+)-ATPase mRNAs. Norepinephrine infusion caused a reduction of Ca(2+)-ATPase mRNA but no change in ryanodine receptor mRNA. There was a corresponding reduction of the immunoreactive Ca(2+)-ATPase protein levels in both heart failure and norepinephrine infusion animals compared to controls. In contrast, the mRNAs of calsequestrin and phospholamban were unchanged in dogs with either congestive heart failure or norepinephrine infusion. Thus, since norepinephrine infusion and congestive heart failure produced similar reductions of Ca(2+)-ATPase mRNA and protein, we postulate that the down-regulation of Ca(2+)-ATPase in congestive heart failure may be caused, at least in part, by sympathetic stimulation that occurs in heart failure.