bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein
Authors: Kurz Mareike , Dalrymple Brian , Wijffels Gene , Kongsuwan Kritaya
Published: May 2004

In
Escherichia coli
, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the
E. coli
Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.
Distinct Mutations in PlcR Explain Why Some Strains of theBacillus cereusGroup Are Nonhemolytic
Authors: Slamti Leyla , Perchat Stéphane , Gominet Myriam , Vilas-Bôas Gislayne , Fouet Agnès , Mock Michèle , Sanchis Vincent , Chaufaux Josette , Gohar Michel , Lereclus Didier
Published: May 2004

Bacillus thuringiensis,Bacillus cereus, andBacillus anthracisare closely related species belonging to theBacillus cereusgroup.B. thuringiensisandB. cereusgenerally produce extracellular proteins, including phospholipases and hemolysins. Transcription of the genes encoding these factors is controlled by the pleiotropic regulator PlcR. Disruption ofplcRinB. cereusandB. thuringiensisdrastically reduces the hemolytic, lecithinase, and cytotoxic properties of these organisms.B. anthracisdoes not produce these proteins due to a nonsense mutation in theplcRgene. We screened 400B. thuringiensisandB. cereusstrains for their hemolytic and lecithinase properties. Eight Hly−Lec−strains were selected and analyzed to determine whether this unusual phenotype was due to a mutation similar to that found inB. anthracis. Sequence analysis of the DNA region including theplcRandpapRgenes of these strains and genetic complementation of the strains with functional copies ofplcRandpapRindicated that different types of mutations were responsible for these phenotypes. We also found that theplcRgenes of threeB. anthracisstrains belonging to different phylogenetic groups contained the same nonsense mutation, suggesting that this mutation is a distinctive trait of this species.
Identification of the Two Missing Bacterial Genes Involved in Thiamine Salvage: Thiamine Pyrophosphokinase and Thiamine Kinase
Authors: Melnick Jonathan , Lis Ewa , Park Joo-Heon , Kinsland Cynthia , Mori Hirotada , Baba Tomoya , Perkins John , Schyns Ghislain , Vassieva Olga , Osterman Andrei , Begley Tadhg P.
Published: May 2004

The genes encoding thiamine kinase in
Escherichia coli
(
ycfN
) and thiamine pyrophosphokinase in
Bacillus subtilis
(
yloS
) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.
Twin frequency and industrial pollution in different regions of Hesse, Germany
Authors: Obi-Osius N , Misselwitz B , Karmaus W , Witten J
Published: May 2004

Aims:To investigate whether twinning occurs more frequently in residents in the vicinity of a toxic waste incinerator (TWI).

Methods:Within a longitudinal environmental study that addressed child health second grade school children and their parents were recruited. The proportion of twinning in the TWI region was compared with two comparison areas. In a second confirmatory investigation, birth records for the years 1994–97 from the Hessian Perinatal Survey (HEPS) were accessed to determine whether the incidence of twinning was higher in regions around the TWI compared to adjacent reference areas.

Results:In the environmental study, 61.5% of the children and 95% of their mothers participated. In mothers, twinning was 5.3% in the TWI region compared to 1.6% and 2.3% in the comparison regions. The proportion of mothers with fertility assessment/treatment was 5.7%, 8.3%, and 0% respectively. The prevalence of twinning was not significantly higher (4.5%) in mothers with treatment compared to mothers without (3.7%). From the HEPS, data of 20 603 births was analysed. The incidence of twins was significantly higher in areas which surround the TWI and other industries (1.4–1.6 per 100 births) compared to births in reference areas (0.8 per 100).

Conclusions:Twinning rates may be associated with exposure to industrial pollution. Future environmental health studies that consider multiple births as an outcome are warranted. These should also investigate whether the incidence of monozygotic or dizygotic twinning may be associated with industrial pollution.
Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves
Authors: Miller Virginia M. , Rodgers George , Charlesworth Jon A. , Kirkland Brenda , Severson Sandra R. , Rasmussen Todd E. , Yagubyan Marineh , Rodgers Jeri C. , Cockerill Franklin R. , Folk Robert L. , Rzewuska-Lech Ewa , Kumar Vivek , Farell-Baril Gerard , Lieske John C.
Published: May 2004

Mechanisms mediating vascular calcification remain incompletely understood. Nanometer scale objects hypothesized to be a type of bacteria (nanobacteria) are associated with calcified geological specimens, human kidney stones, and psammona bodies in ovarian cancer. Experiments were designed to evaluate human vascular tissue for the presence of similar nanometer-scale objects. Calcified human aneurysms ( n = 8), carotid plaques ( n = 2), femoral arterial plaques ( n = 2), and cardiac valves ( n = 2) and noncalcified aneurysms from patients with bicuspid aortic valve disease ( n = 2) were collected as surgical waste from the Heart Hospital of Austin, Austin, Texas, and Mayo Clinic, Rochester, Minnesota. Whole mounts or adjacent sections from each specimen were examined by electron microscopy, stained for calcium phosphate, or stained with a commercially available antibody (8D10). Filtered (0.2 μm) homogenates of aneurysms were cultured and costained with 8D10 antibody followed by PicoGreen to detect DNA or incubated with [3H]uridine. Staining for calcium phosphate was heterogeneously distributed within all calcified tissues. Immunological staining with 8D10 was also heterogeneously distributed in areas with and without calcium phosphate. Analysis of areas with positive immunostaining identified spheres ranging in size from 30 to 100 nm with a spectral pattern of calcium and phosphorus (high-energy dispersive spectroscopy). Nanosized particles cultured from calcified but not from noncalcified aneurysms were recognized by a DNA-specific dye and incorporated radiolabeled uridine, and, after decalcification, they appeared via electron microscopy to contain cell walls. Therefore, nanometer-scale particles similar to those described as nanobacteria isolated from geological specimens and human kidney stones can be visualized in and cultured from calcified human cardiovascular tissue.
Degradation of rat cardiac troponin I during ischemia independent of reperfusion
Authors: Palmer Brian S. , Klawitter Paul F. , Reiser Peter J. , Angelos Mark G.
Published: May 2004

Cardiac troponin I (cTnI) degradation has been noted in the stunned myocardium of rodents after ischemia and reperfusion and is one proposed mechanism for the decreased left ventricular (LV) contractility in postischemic hearts. cTnI degradation has been best described after reperfusion of the ischemic myocardium. The effect of ischemia, independent of reperfusion, on cTnI breakdown has not been well characterized. We tested the hypothesis that progressive cTnI degradation occurs with increasing durations of ischemia and that this ischemia-based degradation is, in part, oxidant mediated. Isolated perfused rat hearts underwent global ischemia of 15, 20, or 25 min with and without reperfusion. A second series of hearts was treated with the antioxidants tiron (10 mM) and N-acetylcysteine (4 mM) before 20 min of global ischemia without reperfusion. cTnI degradation was measured using a cTnI-specific antibody and Western blot analyses. A progressive increase in cTnI degradation was seen with increasing duration of ischemia (no reperfusion), which correlated with the return of LV developed pressure during reperfusion. The extent of cTnI degradation was increased in hearts pretreated with antioxidants, although the qualitative degradation pattern was not altered. We conclude that a time-dependent cTnI breakdown occurs during global ischemia that is independent of reperfusion. cTnI breakdown during ischemia is further increased in the presence of antioxidants, suggesting ROS generated during ischemia may play a cTnI protective role.
Zero‐dispersion limit for integrable equations on the half‐linewith linearisable data
Authors: Fokas A. S. , Kamvissis S.
Published: May 2004

We study the zero‐dispersion limit for certain initial boundary
value problems for the defocusing nonlinear Schrödinger
(NLS) equationand for the Korteweg‐de Vries
(KdV)equation with dominant surface tension. These
problems are formulated on the half‐line and they involve
linearisable boundaryconditions.
Promoter Analysis of Human Corticotropin-Releasing Factor (CRF) Type 1 Receptor and Regulation by CRF and Urocortin
Authors: Parham Kelly L. , Zervou Sevasti , Karteris Emmanouil , Catalano Rob D. , Old Robert W. , Hillhouse Edward W.
Published: May 2004

We report the full genomic organization of the human gene for the corticotropin-releasing factor (CRF) receptor type 1 (CRFR1), with complete mapping of exons 1–14. The 5′ flanking region (2.4 kb) of the gene encoding for human CRFR1 was isolated, sequenced, and characterized. Two major transcriptional start sites were determined at −265 and −238, relative to the ATG start site (+1). Transient expression of constructs containing sequentially deleted 5′-flanking sequences of CRFR1 fused to luciferase, revealed the minimal promoter sequence 370 bp in size, as shown by assays in neuroblastoma (SH-5YSY), teratocarcinoma (NT2), and adenocarcinoma (MCF 7) cell lines. CRF and UCN markedly increased promoter activity during transient CRFR1 expression studies. Similarly, CRF and UCN up-regulate the endogenous CRFR1 at the mRNA level in NT2 and MCF 7 cells. To dissect further the mechanisms involved, we have used primary myometrial cells transfected with the CRFR1 promoter. CRF and UCN increased the promoter activity, an effect blocked by protein kinase (PK)A and PKC inhibitors. Both CRF and UCN cause a positive feedback effect in primary cultures of human pregnant myometrial cells, by increasing mRNA expression of CRFR1. This effect appears to be dependent on activation of both PKA and PKC by CRF, whereas UCN′s effect was mediated solely via PKC activation. Collectively, our data suggest that the CRFR1 gene is under the influence of both CRF and UCN, acting via distinct signaling pathways to create a positive feedback loop and regulate further the transcription of the receptor.
Economic Consequences of Unused Medications in Houston, Texas
Authors: Garey Kevin W , Johle Mikki L , Behrman Kathy , Neuhauser Melinda M
Published: May 2004

BACKGROUND:

It is likely that a large amount of unused and outdated medications exists in households throughout the US; however, the amount and potential costs of these medications are unknown.

OBJECTIVE:

To determine the amount, types, and costs of unused medications present in a neighborhood surrounding a community pharmacy in Houston, Texas.

METHODS:

A community trial was conducted between April and September 2002. This pilot study investigated the quantity and types of drugs returned to a community pharmacy over a 6-month period.

RESULTS:

During the study period, approximately 17 000 oral pills worth over $26 000 were collected in 1315 medication containers. Medications collected were from all drug classes and types (pharmaceutical samples, over-the-counter and prescription drugs).

CONCLUSIONS:

This study demonstrated that an enormous amount of unused medications were present in a community in the US. Community pharmacies may be an ideal venue to collect and destroy these unused drugs.
CHINA'S HISTORY ACTIVISTS AND THE WAR OF RESISTANCE AGAINST JAPAN: History in the Making
Authors: Reilly James
Published: May 2004

In China over the past two decades, a group of “history activists” has maximized its professional independence, social credibility, academic resources, and international connections to usurp many traditional roles of the state in building new institutions and engaging in activism aimed at documenting and disseminating information on Japan's wartime atrocities.
Clinical assessment of behavioral coping responses: preliminary results from a brief inventory
Authors: McCracken Lance M. , Eccleston Christopher , Bell Louise
Published: May 2004

Patients and clinicians sometimes take coping with chronic pain primarily as a process of gaining more control over pain. An alternate approach might include helping the pain sufferer to discriminate parts of their situation that can be effectively controlled from those that cannot. When faced with situations that do not yield to attempts at direct control patients may gain better results from leaving those situations as they are and investing their efforts elsewhere. This study was designed to examine this type of expanded view of coping with pain, a view that includes both attempts at control and acceptance. 200 adults seeking treatment for chronic pain were the subjects of this investigation. They completed a number of self‐report inventories including a measure called the Brief Pain Coping Inventory, an inventory assessing accepting responses to pain as well as pain management responses standardly targeted by cognitive‐behavioral treatment methods. Preliminary results showed that the BPCI yields scores with adequate temporal consistency and validity. Further results showed that a number of the responses assessed by the BPCI were reliable predictors of patient functioning. In general less frequent struggling to control pain, fewer palliative and avoidant coping responses, and more explicit persistence with activity despite acknowledged pain were associated with less depression and anxiety and greater life functioning. These results demonstrate that, in some instances, attempts at avoidance and control of chronic pain may be less helpful compared with a willingness to experience pain and focus on functioning.
Inhibition of the gamma interferon response by a Sendai virus C protein mutant with no STAT1‐binding ability
Authors: Gotoh Bin , Takeuchi Kenji , Komatsu Takayuki
Published: May 2004

Sendai virus C protein interacts with the signal transducer and activator of transcription (STAT) 1. This interaction is believed to be essential for the Sendai virus inhibition of the interferon (IFN) response. We here analyzed CF170S (a C protein mutant with the F170S mutation) with no STAT1‐binding ability. CF170S lacked the ability to inhibit the IFN‐α response, but retained the ability to inhibit the IFN‐γ response. IFN‐γ stimulation caused STAT1 phosphorylation, formation of the gamma‐activated factor capable of binding to a gamma‐activated sequence DNA probe, and STAT1 nuclear translocation, even in the presence of CF170S. These results suggest that C protein has the STAT1‐binding‐independent anti‐IFN‐γ mechanism, which targets processes after the STAT1 nuclear translocation event.
Hypoxia‐dependent regulation of PHD1: cloning and characterization of the human PHD1/EGLN2 gene promoter
Authors: Erez Neta , Stambolsky Perry , Shats Igor , Milyavsky Michael , Kachko Tami , Rotter Varda
Published: May 2004

The recent identification of hypoxia‐inducible‐factor (HIF) prolyl hydroxylases (PHD1, 2, and 3), which modify HIF‐1α in an oxygen‐dependent manner, provided an important link between oxygen availability and hypoxia‐induced gene expression. However, little is known about the regulation of the PHDs. To investigate the transcriptional regulation of PHD1, we cloned the PHD1 gene promoter. Here, we report that the expression of PHD1 is reduced under hypoxic conditions. Furthermore, we identified binding sites for aryl hydrocarbon nuclear translocator (ARNT/HIF‐1β) within the PHD1 promoter, and showed that ARNT is associated in vivo with the PHD1 promoter following hypoxia, which implies a role for ARNT in the hypoxia‐dependent regulation of PHD1. Taken together, our findings suggest a hypoxia‐induced regulatory loop of PHD1 expression, mediated by ARNT.
Crystal structure of Rab9 complexed to GDP reveals a dimer with an active conformation of switch II
Authors: Wittmann Julia G , Rudolph Markus G
Published: May 2004

The small GTPase Rab9 is an essential regulator of vesicular transport from the late endosome to the trans‐Golgi network, as monitored by the redirection of the mannose‐6‐phosphate receptors. The crystal structure of Rab9 complexed to GDP, Mg2+, and Sr2+ reveals a unique dimer formed by an intermolecular β‐sheet that buries the switch I regions. Surface area and shape complementarity calculations suggest that Rab9 dimers can form an inactive, membrane‐bound pool of Rab9 · GDP that is independent of GDI. Mg2+‐bound Rab9 represents an inactive state, but Sr2+‐bound Rab9 · GDP displays activated switch region conformations, mimicking those of the GTP state. A hydrophobic tetrad is formed resembling an effector‐discriminating epitope found only in GTP‐bound Rab proteins.
Bringing the party back in: the CCP and the trajectory of market transition in China
Authors: Shevchenko Alexei
Published: May 2004

The article suggests a conceptual framework for understanding the logic and dynamics of the Chinese Communist Party’s (CCP’s) organizational adaptation to market conditions and the consequences of this process for the trajectory and outcomes of the Chinese reform process. I define the CCP’s adaptation to market as ‘‘entrepreneurial’’ in nature with party cadres and agencies at different levels becoming not only oriented towards the goal of increasing local economic output and productivity but also directly involved in profit-making activities. I also argue that despite increased opportunities for rent-seeking and corruption, the CCP’s struggle for its organizational survival in the process of entrepreneurial adaptation has positively influenced the trajectory of Chinese market transition. By providing its agents powerful incentives to uphold the necessary degree of compliance with central policies, by limiting lucrative ‘‘easy avenues’’ for profit-seeking and value-subtracting behavior, by preventing the formation of powerful interests capable of hijacking the reform process, and by imposing painful but necessary economic rationalization measures, the party became an imperfect but crucial substitute for the lack of institutions necessary to guide the process of creating markets.
Why politicians want constitutional courts: the Russian case
Authors: Thorson Carla
Published: May 2004

Why are judicial review mechanisms being incorporated into so many democratizing states? This study analyzes why politicians create an independent judicial institution with the authority to overrule their own decisions. It sheds light on the role constitutional courts play in the consolidation phase of a democratic transition, focusing on one of those countries with no tradition of independent judicial review or of democratic forms of governance—Russia. Past practices and historical precedent do not support the formation of an independent judiciary in Russia, and yet a potentially powerful constitutional court now exists. Moreover, during the course of the transition from the Soviet state to the Russian Republic, there were three attempts to create an independent judicial review mechanism only one of which could be termed a success. This analysis focuses on the self-interested calculations of politicians in forming each of these three institutions, demonstrating that political actors establish a constitutional court to enhance their democratic credibility.
Protein-lipid interactions and phosphoinositide metabolism in membrane traffic: Insights from vesicle recycling in nerve terminals
Authors: Wenk Markus R. , De Camilli Pietro
Published: May 2004

Great progress has been made in the elucidation of the function of proteins in membrane traffic. Less is known about the regulatory role of lipids in membrane dynamics. Studies of nerve terminals, compartments highly specialized for the recycling of synaptic vesicles, have converged with studies from other systems to reveal mechanisms in protein-lipid interactions that affect membrane shape as well as the fusion and fission of vesicles. Phosphoinositides have emerged as major regulators of the binding of cytosolic proteins to the bilayer. Phosphorylation on different positions of the inositol ring generates different isomers that are heterogeneously distributed on cell membranes and that together with membrane proteins generate a “dual keys” code for the recruitment of cytosolic proteins. This code helps controlling vectoriality of membrane transport. Powerful methods for the detection of lipids are rapidly advancing this field, thus complementing the broad range of information about biological systems that can be obtained from genomic and proteomic approaches.
Identification of Ald6p as the target of a class of small-molecule suppressors of FK506 and their use in network dissection
Authors: Butcher Rebecca A. , Schreiber Stuart L.
Published: May 2004

FK506 inhibits the Ca
2+
/calmodulin-dependent protein phosphatase calcineurin, which plays a critical role in yeast subjected to salt stress. A chemical genetic screen for small molecules that suppress growth inhibition by high NaCl plus FK506 identified a structurally related class of suppressors of FK506 (SFKs) named SFKs 2–4. To identify possible protein targets for these small molecules, a genome-wide screen of ≈4,700 haploid yeast deletion strains was undertaken for strains showing resistance to high NaCl plus FK506. This screen yielded a number of genes not previously implicated in salt stress, including
ALD6
, which encodes an NADP
+
-dependent aldehyde dehydrogenase, and
UTR1
, which encodes an NAD
+
kinase. Transcriptional profiling of yeast treated with SFK2 indicated that the SFKs target the Ald6p pathway. In addition, screening of the deletion strains for hypersensitivity to SFK2 yielded
ZWF1
, encoding glucose-6-phosphate dehydrogenase, which has been shown to play an overlapping role with Ald6p in NADPH production. Furthermore, the SFKs inhibited the activity of Ald6p
in vitro
. Having established that the SFKs target Ald6p, they were used as tools to implicate systematically other gene products in the Ald6p pathway, including Utr1p, which may function by supplying Ald6p with its NADP
+
cofactor. Furthermore, growth improvement by the SFKs on high NaCl plus FK506 was shown to require
GPD1
, which encodes an NADH-dependent glycerol-3-phosphate dehydrogenase that is important for the production of glycerol in response to osmotic stress.
RETRACTED: Suppression of pathogen-inducible NO synthase (iNOS) activity in tomato increases susceptibility to
Pseudomonas syringae
Authors: Chandok Meena R. , Ekengren Sophia K. , Martin Gregory B. , Klessig Daniel F.
Published: May 2004

Inducible NO synthase (iNOS) activity is induced upon pathogen inoculation in resistant, but not susceptible, tobacco and
Arabidopsis
plants. It was shown recently that a variant form of the
Arabidopsis
P protein (AtvarP) has iNOS activity. P protein is part of the glycine decarboxylase complex (GDC). It is unclear whether P protein also has iNOS activity and, if so, whether AtvarP, P, or both, play a role in plant defense. Here, we show that iNOS activity is induced in both resistant and susceptible tomato leaves upon inoculation with the
Pseudomonas syringae
pv.
tomato
strain DC3000. Virus-induced gene-silencing targeting
LevarP
, a putative tomato ortholog of
AtvarP
, led to complete suppression of DC3000-induced iNOS activation and an ≈80% reduction in GDC activity; it also increased disease-symptom severity and DC3000 growth in both resistant and susceptible tomato. To determine whether enhanced susceptibility exhibited by
LevarP
-silenced, susceptible tomato was due to loss of (
i
) iNOS activity, (
ii
) GDC activity, or (
iii
) both, GDC activity was inhibited with or without concurrent suppression of iNOS. Treatment with methotrexate inhibited both iNOS and GDC activities and resulted in increased susceptibility, comparable with that observed in
LevarP
-silenced plants. When normal iNOS activity was maintained in the presence of methotrexate by the addition of tetrahydrobiopterin, there was no change in susceptibility, despite a dramatic reduction in GDC activity. Together, these results indicate that iNOS contributes to host defense response against DC3000.
The total length of spindle microtubules depends on the number of chromosomes present.
Authors: Nicklas R B , Gordon G W
Published: May 2004

We extracted chromosomes by micromanipulation from Melanoplus differentialis spermatocytes, producing metaphase spindles with only one or a few chromosomes instead of the usual complement of 23. Cells with various numbers of chromosomes were prepared for electron microscopy, and spindle microtubule length was measured. A constant increment of microtubule length was lost upon the removal of each chromosome; we estimate that only approximately 40% of the original length would remain in the total absence of chromosomes. Unexpectedly, kinetochore microtubules were not the only ones affected when chromosomes were removed: nonkinetochore microtubules accounted for a substantial fraction of the total length lost. No compensatory increase in microtubule length outside the spindle was found. Studies by others show that the kinetochore microtubules of extracted chromosomes are left behind in the cell and dissassemble. The resulting increase in subunit concentration would be expected from in vitro studies to drive microtubule assembly until the original total microtubule length was restored, but that did not happen in these living cells. We conclude that the assembly of a certain, large fraction of microtubule subunits into stable microtubules is dependent on the presence of chromosomes. Possible explanations include (a) limits on microtubule length that prevent any net assembly of the subunits released after chromosomes are removed or (b) a promotion of microtubule assembly by chromosomes, which therefore is reduced in their absence. Chromosome-dependent regulation of microtubule length may account for some features of normal mitosis.
Uptake of chemically modified low density lipoproteins in vivo is mediated by specific endothelial cells.
Authors: Pitas R E , Boyles J , Mahley R W , Bissell D M
Published: May 2004

Acetoacetylated (AcAc) and acetylated (Ac) low density lipoproteins (LDL) are rapidly cleared from the plasma (t1/2 approximately equal to 1 min). Because macrophages, Kupffer cells, and to a lesser extent, endothelial cells metabolize these modified lipoproteins in vitro, it was of interest to determine whether endothelial cells or macrophages could be responsible for the in vivo uptake of these lipoproteins. As previously reported, the liver is the predominant site of the uptake of AcAc LDL; however, we have found that the spleen, bone marrow, adrenal, and ovary also participate in this rapid clearance. A histological examination of tissue sections, undertaken after the administration of AcAc LDL or Ac LDL (labeled with either 125I or a fluorescent probe) to rats, dogs, or guinea pigs, was used to identify the specific cells binding and internalizing these lipoproteins in vivo. With both techniques, the sinusoidal endothelial cells of the liver, spleen, bone marrow, and adrenal were labeled. Less labeling was noted in the ovarian endothelia. Uptake of AcAc LDL by endothelial cells of the liver, spleen, and bone marrow was confirmed by transmission electron microscopy. These data suggest uptake through coated pits. Uptake of AcAc LDL was not observed in the endothelia of arteries (including the coronaries and aorta), veins, or capillaries of the heart, testes, kidney, brain, adipose tissue, and duodenum. Kupffer cells accounted for a maximum of 14% of the 125I-labeled AcAc LDL taken up by the liver. Isolated sinusoidal endothelial cells from the rat liver displayed saturable, high affinity binding of AcAc LDL (Kd = 2.5 X 10(-9) M at 4 degrees C), and were shown to degrade AcAc LDL 10 times more effectively than aortic endothelial cells. These data indicate that specific sinusoidal endothelial cells, not the macrophages of the reticuloendothelial system, are primarily responsible for the removal of these modified lipoproteins from the circulation in vivo.
Immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells.
Authors: Roth J , Lentze M J , Berger E G
Published: May 2004

Galactosyltransferase immunoreactive sites were localized in human duodenal enterocytes by the protein A-gold technique on thin sections from low temperature Lowicryl K4M embedded biopsy specimens. Antigenic sites detected with affinity-purified, monospecific antibodies were found at the plasma membrane of absorptive enterocytes with the most intense labeling appearing along the brush border membrane. The lateral plasma membrane exhibited a lower degree of labeling at the level of the junctional complexes but the membrane interdigitations were intensely labeled. The labeling intensity decreased progressively towards the basal part of the enterocytes and reached the lowest degree along the basal plasma membrane. Quantitative evaluation of the distribution of gold-particle label proved its preferential orientation to the outer surface of the plasma membrane. In addition to this membrane-associated labeling, the glycocalyx extending from the microvillus tips was heavily labeled. Occasionally, cells without plasma membrane labeling were found adjacent to positive cells. The demonstration of ecto-galactosyltransferase on membranes other than Golgi membranes precludes its general use as a marker for Golgi membrane fractions. The possible function of galactosyltransferase on a luminal plasma membrane is unclear at present, but a role in adhesion appears possible on the basolateral plasma membrane.
Localization of tektin filaments in microtubules of sea urchin sperm flagella by immunoelectron microscopy.
Authors: Linck R W , Amos L A , Amos W B
Published: May 2004

Extraction of doublet microtubules from the sperm flagella of the sea urchin Strongylocentrotus purpuratus with sarkosyl (0.5%)-urea (2.5 M) yields a highly pure preparation of "tektin" filaments that we have previously shown to resemble intermediate filament proteins. They form filaments 2-3 nm in diameter as seen by negative stain electron microscopy and are composed of approximately equal amounts of three polypeptide bands with apparent molecular weights of 47,000, 51,000, and 55,000, as determined by SDS PAGE. We prepared antibodies to this set of proteins to localize them in the doublet microtubules of S. purpuratus and other species. Tektins and tubulin were antigenically distinct when tested by immunoblotting with affinity-purified antitektin and antitubulin antibodies. Fixed sperm or axonemes from several different species of sea urchin showed immunofluorescent staining with antitektin antibodies. We also used antibodies coupled to gold spheres to localize the proteins by electron microscopy. Whereas a monoclonal antitubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol. 93:576-582) decorates intact microtubules along their lengths, antitektins labeled only the ends of intact microtubules and sarkosyl-insoluble ribbons. However, if microtubules and ribbons attached to electron microscope grids were first extracted with sarkosyl-urea, the tektin filaments that remain were decorated by antitektin antibodies throughout their length. These results suggest that tektins form integral filaments of flagellar microtubule walls, whose antigenic sites are normally masked, perhaps by the presence of tubulin around them.
Chronic denervation of rat hemidiaphragm: maintenance of fiber heterogeneity with associated increasing uniformity of myosin isoforms.
Authors: Carraro U , Morale D , Mussini I , Lucke S , Cantini M , Betto R , Catani C , Dalla Libera L , Danieli Betto D , Noventa D
Published: May 2004

During several months of denervation, rat mixed muscles lose slow myosin, though with variability among animals. Immunocytochemical studies showed that all the denervated fibers of the hemidiaphragm reacted with anti-fast myosin, while many reacted with anti-slow myosin as well. This has left open the question as to whether multiple forms of myosin co-exist within individual fibers or a unique, possibly embryonic, myosin is present, which shares epitopes with fast and slow myosins. Furthermore, one can ask if the reappearance of embryonic myosin in chronically denervated muscle is related both to its re-expression in the pre-existing fibers and to cell regeneration. To answer these questions we studied the myosin heavy chains from individual fibers of the denervated hemidiaphragm by SDS PAGE and morphologically searched for regenerative events in the long term denervated muscle. 3 mo after denervation the severely atrophic fibers of the hemidiaphragm showed either fast or a mixture of fast and slow myosin heavy chains. Structural analysis of proteins sequentially extracted from muscle cryostat sections showed that slow myosin was still present 16 mo after denervation, in spite of the loss of the selective distribution of fast and slow features. Therefore muscle fibers can express adult fast myosin not only when denervated during their differentiation but also after the slow program has been expressed for a long time. Light and electron microscopy showed that the long-term denervated muscle maintained a steady-state atrophy for the rat's life span. Some of the morphological features indicate that aneural regeneration events continuously occur and significantly contribute to the increasing uniformity of the myosin gene expression in long-term denervated diaphragm.
Rod cells dissociated from mature salamander retina: ultrastructure and uptake of horseradish peroxidase.
Authors: Townes-Anderson E , MacLeish P R , Raviola E
Published: May 2004

To test the effects of isolation on adult neurons, we investigated the fine structure and synaptic activity of rod cells dissociated from the mature salamander retina and maintained in vitro. First, freshly isolated rod cells appeared remarkably similar to their counterparts in the intact retina: the outer segment retained its stack of membranous disks and the inner segment contained its normal complements of organelles. Some reorganization of the cell surface, however, was observed: (a) radial fins, present at the level of the cell body, were lost; and (b) the apical and distal surfaces of the inner and outer segments, respectively became broadly fused. Second, the synaptic endings or pedicles retained their presynaptic active zones: reconstruction of serially sectioned pedicles by using three-dimensional computer graphics revealed that 73% of the synaptic ribbons remained attached to the plasmalemma either at the cell surface or along its invaginations. Finally, tracer experiments that used horseradish peroxidase demonstrated that dissociated rod cells recycled synaptic vesicle membrane in the dark and thus probably released transmitter by exocytosis. Under optimal conditions, a maximum of 40% of the synaptic vesicles within the pedicle were labeled. As in the intact retina, uptake of horseradish peroxidase was suppressed by light. Thus, freshly dissociated receptor neurons retained many of their adult morphological and physiological characteristics. In long-term culture, the photoreceptors tended to round up; however, active zones were present even 2 wk after removal of the postsynaptic processes.
Glycoprotein 115, a glycoprotein isolated from chick blood vessels, is widely distributed in connective tissue.
Authors: Colombatti A , Bressan G M , Castellani I , Volpin D
Published: May 2004

An extracellular glycoprotein (gp 115) with an apparent Mr = 115,000 isolated from chick aortas (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin, 1983, J. Biol. Chem., 258:13262-13267), was used to immunize mice. The antisera were shown to specifically recognize gp 115 by numerous criteria: a major band around Mr = 115,000 plus minor bands of lower Mr were visible by immunoblotting on aorta extracts, and a similar pattern was observed with a monoclonal antibody; no cross-reactivity was detected by radioimmunobinding with other extracellular proteins, namely, fibronectin, laminin, and collagen types I, III, IV, V, and VI. Antigen distribution on frozen tissue sections from newborn chicks was investigated by using affinity-purified antibody. Strong immunoreactivity was always found in blood vessels. In the digestive tract, the fluorescent staining was localized both at the level of muscular layers and in the stromal matrix of the villi. Within skeletal muscle and myocardium, staining was associated with large connective tissue bundles and the matrix around each muscle fiber. Intense fluorescence was observed in the kidney, in smooth muscle cells rich areas of parabronchi, and within the portal space and along liver sinusoids. The antigen was not detected at the epidermal-dermal junction; immunoreactivity in the dermis was present as a diffuse fibrillar pattern. That the antigen detected by immunofluorescence in the various organs was indeed gp 115 was demonstrated by immunoblotting analysis: as in aorta extracts, a major band around Mr = 115,000 was detected in several tissues. Antibody-reacting material was also incorporated into the extracellular matrix produced by embryo smooth muscle cells grown in vitro and was organized as a meshwork of fine fibrils.
Analytical study of microsomes and isolated subcellular membranes from rat liver. IX. Nicotinamide adenine dinucleotide glycohydrolase: a plasma membrane enzyme prominently found in Kupffer cells.
Authors: Amar-Costesec A , Prado-Figueroa M , Beaufay H , Nagelkerke J F , van Berkel T J
Published: May 2004

The distribution of nicotinamide adenine dinucleotide (NAD) glycohydrolase in rat liver was investigated by subcellular fractionation and by isolation of hepatocytes and sinusoidal cells. The behavior of NAD glycohydrolase in subcellular fractionation was peculiar because, although the enzyme was mainly microsomal, plasma membrane preparations contained distinctly more NAD glycohydrolase than could be accounted for by their content in elements derived from the endoplasmic reticulum or the Golgi complex identified by glucose-6-phosphatase and galactosyltransferase, respectively. When microsomal and plasmalemmal preparations were brought to equilibrium in a linear-density gradient, NAD glycohydrolase differed from these enzymes and behaved like 5'-nucleotidase and alkaline phosphodiesterase I. NAD glycohydrolase was markedly displaced towards higher densities after treatment with digitonin. This behavior in density-gradient centrifugation strongly suggests that NAD glycohydrolase is an exclusive enzyme of the plasma membrane. NAD glycohydrolase differed clearly from other plasmalemmal enzymes when the liver was fractionated into hepatocytes and sinusoidal cells; its specific activity was considerably greater in sinusoidal cell than in hepatocyte preparations. Further subfractionation of sinusoidal cell preparations into endothelial and Kupffer cells by counterflow elutriation showed that NAD glycohydrolase is more active in Kupffer cells. We estimate that the specific activity of NAD glycohydrolase activity is at least 65-fold higher at the periphery of Kupffer cells than at the periphery of hepatocytes. As the enzyme shows not structure-linked latency and is an exclusive constituent of the plasma membranes, we conclude that it is an ectoenzyme that cannot lead to a rapid turnover of the cytosolic pyridine nucleotides.
Structure of C protein purified from cardiac muscle.
Authors: Hartzell H C , Sale W S
Published: May 2004

C protein is a component of the thick filament of striated muscles. Although the function of C protein remains unknown, a variety of evidence suggests that C protein may regulate actin-myosin interaction or be involved in structural support or elasticity of the sarcomere. We have previously proposed (Hartzell, H. C., 1984, J. Gen. Physiol., 83:563-588) that C protein is involved in regulating twitch relaxation in cardiac muscle. To gain further insight into the function of C protein, we have studied the structure of C protein purified from chicken heart. C protein was purified from extracts of detergent-washed myofibrils by sequential hydroxylapatite and DEAE-Sephacel chromatography. C protein was judged greater than 95% pure by SDS PAGE. The polypeptide subunit had a molecular weight of 155,000 and the native molecule sedimented on linear sucrose or glycerol gradients at 4-5S. For electron microscopy, purified C protein was dialyzed and diluted into a volatile buffer in 50% glycerol, aspirated onto mica, dried under vacuum, and rotary platinum-shadowed. Replicas revealed particles of relatively homogeneous overall dimensions. Over half of the particles were V-shaped. The "arm" lengths of the V-shaped particles were 22 +/- 4.5 nm (SD). Gel filtration on Sephacryl S-300 demonstrated that purified C protein had a Stokes' radius of 5.07 nm. Measurements of viscosity gave an intrinsic viscosity of 16.5 cm3/g. These data are consistent with the electron microscopic data and suggest that C protein in heart muscle is asymmetric. The C protein molecule is large enough to extend from the surface of a thick filament to adjacent thin or thick filaments.
Functional determinants in transit sequences: import and partial maturation by vascular plant chloroplasts of the ribulose-1,5-bisphosphate carboxylase small subunit of Chlamydomonas.
Authors: Mishkind M L , Wessler S R , Schmidt G W
Published: May 2004

The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved.
Giant axonal neuropathy: a conditional mutation affecting cytoskeletal organization.
Authors: Klymkowsky M W , Plummer D J
Published: May 2004

Giant axonal neuropathy (GAN) results from autosomal recessive mutations (gan-) that affect cytoskeletal organization; specifically, intermediate filaments (IFs) are found collapsed into massive bundles in a variety of different cell types. We studied the gan- fibroblast lines WG321 and WG139 derived from different GAN patients. Although previous studies implied that the gan- IF phenotype was constitutive, we find that it is conditional. That is, when cells were grown under the permissive condition of medium containing over 2% fetal calf serum, most cells had normal IF organization. IF bundles formed when gan- cells were transferred to the nonpermissive condition of low (0.1%) serum. Microtubule organization appeared normal in the presence or absence of serum. The effect of serum starvation was largely blocked or reversed by the addition of BSA to the culture media. We found no evidence that the gan- phenotype depends upon progress through the cell cycle. We discuss the possible role of serum effects in the etiology of GAN and speculate as to the molecular nature of the gan- defect.
Characterization of a myosin heavy chain in the conductive system of the adult and developing chicken heart.
Authors: González-Sánchez A , Bader D
Published: May 2004

A monoclonal antibody (anterior latissimus dorsi 58 [ALD58]; antimyosin heavy chain, MHC) directed against myosin from slow tonic muscle was found to react specifically with the striated muscle cells of the conductive system in the adult chicken heart. This monoclonal antibody was used to study the expression of myosin in the conductive system of the adult and developing heart. Using immunofluorescence microscopy with ALD58, muscle cells of the conductive system were demonstrated in both the atria and ventricles of the adult heart as previously shown by Sartore et al. (Sartore, S., S. Pierobon-Bormioli, and S. Schiafinno, 1978, Nature (Lond.), 274: 82-83). Radioactive myosin from adult atria and ventricles was precipitated with ALD58 and subjected to limited proteolysis and subsequent peptide mapping. Peptide maps of ALD58 reactive myosin from atria and ventricles were very similar, if not identical, but differed from peptide maps of ordinary atrial and ventricular myosin. The same antibody was used to study cardiac myogenesis in the chick embryo. When ALD58 was reacted with myosin isolated from atria and ventricles at selected stages of development in radioimmunoassays, reactivity was not observed until the last week of embryonic life (greater than 15 d of egg incubation). Thereafter concomitant and progressively increased reactivity was observed in atrial and ventricular preparations. Also, no ALD58 positive cells were observed in immunofluorescence studies of embryonic hearts until 17 d of egg incubation. Primary cell cultures of embryonic hearts also proved to be negative for this antibody. This study demonstrates that an epitope recognized by ALD58 associated with an antimyosin heavy chain of striated muscle cells of the adult heart conductive system is absent or present in only small amounts in the early embryonic heart.
Purification of brain tubulin-tyrosine ligase by biochemical and immunological methods.
Authors: Schröder H C , Wehland J , Weber K
Published: May 2004

Tubulin-tyrosine ligase (TTL), the enzyme responsible for the reversible addition of a tyrosine residue at the carboxyl end of alpha-tubulin, has been purified from porcine brain using a purification scheme based on standard biochemical procedures. The enzyme preparation was nearly homogeneous (purity greater than 95%), was free of tubulin, and could be stored in the presence of glycerol for several months without loss in activity. To develop a more convenient purification of TTL, we have isolated mouse hybridoma cells secreting antibodies to TTL. These monoclonal antibodies recognize TTL not only in brain tissue but also in the liver of various mammals. Monoclonal antibodies isolated from ascites fluid allowed a rapid purification of TTL from a crude brain extract. TTL stayed bound to the immunoaffinity column in 1.5 M NaCl and was eluted with 3 M MgCl2. Highly active TTL was recovered nearly quantitatively at greater than 95% purity and could be stabilized in the presence of glycerol. Glycerol gradient centrifugation, SDS gel electrophoresis and immunoblots identified TTL as a monomeric protein with an apparent polypeptide molecular weight of about 40,000. A one to one complex of TTL with alpha beta-tubulin was observed by gradient centrifugation.