bluuebunny/crossref_metadata_2025 huggingface dataset:
Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco
Authors: Huang Chun Y. , Ayliffe Michael A. , Timmis Jeremy N.
Published: June 2004

Transfer of organelle DNA into the nuclear genome has been significant in eukaryotic evolution, because it appears to be the origin of many nuclear genes. Most studies on organelle DNA transfer have been restricted to evolutionary events but experimental systems recently became available to monitor the process in real time. We designed an experimental screen to detect plastid DNA (ptDNA) transfers to the nucleus in whole plants grown under natural conditions. The resultant genotypes facilitated investigation of the evolutionary mechanisms underlying ptDNA transfer and nuclear integration. Here we report the characterization of nuclear loci formed by integration of newly transferred ptDNA. Large, often multiple, fragments of ptDNA between 6.0 and 22.3 kb in size are incorporated into chromosomes at single Mendelian loci. The lack of chloroplast transcripts of comparable size to the ptDNA integrants suggests that DNA molecules are directly involved in the transfer process. Microhomology (2–5 bp) and rearrangements of ptDNA and nuclear DNA were frequently found near integration sites, suggesting that nonhomologous recombination plays a major role in integration. The mechanisms of ptDNA integration appear similar to those of biolistic transformation of plant cells, but no sequence preference was identified near junctions. This article provides substantial molecular analysis of real-time ptDNA transfer and integration that has resulted from natural processes with no involvement of cell injury, infection, and tissue culture. We highlight the impact of cytoplasmic organellar genome mobility on nuclear genome evolution.
Computational inference of scenarios for α-proteobacterial genome evolution
Authors: Boussau Bastien , Karlberg E. Olof , Frank A. Carolin , Legault Boris-Antoine , Andersson Siv G. E.
Published: June 2004

The α-proteobacteria, from which mitochondria are thought to have originated, display a 10-fold genome size variation and provide an excellent model system for studies of genome size evolution in bacteria. Here, we use computational approaches to infer ancestral gene sets and to quantify the flux of genes along the branches of the α-proteobacterial species tree. Our study reveals massive gene expansions at branches diversifying plant-associated bacteria and extreme losses at branches separating intracellular bacteria of animals and humans. Alterations in gene numbers have mostly affected functional categories associated with regulation, transport, and small-molecule metabolism, many of which are encoded by paralogous gene families located on auxiliary chromosomes. The results suggest that the α-proteobacterial ancestor contained 3,000 -5,000 genes and was a free-living, aerobic, and motile bacterium with pili and surface proteins for host cell and environmental interactions. Approximately one third of the ancestral gene set has no homologs among the eukaryotes. More than 40% of the genes without eukaryotic counterparts encode proteins that are conserved among the α-proteobacteria but for which no function has yet been identified. These genes that never made it into the eukaryotes but are widely distributed in bacteria may represent bacterial drug targets and should be prime candidates for future functional characterization.
Learning problems, delayed development, and puberty
Authors: Wright Beverly A. , Zecker Steven G.
Published: June 2004

Language-based learning disorders such as dyslexia affect millions of people, but there is little agreement as to their cause. New evidence from behavioral measures of the ability to hear tones in the presence of background noise indicates that the brains of affected individuals develop more slowly than those of their unaffected counterparts. In addition, it seems that brain changes occurring at ≈10 years of age, presumably associated with puberty, may prematurely halt this slower-than-normal development when improvements would normally continue into adolescence. The combination of these ideas can account for a wide range of previous results, suggesting that delayed brain development, and its interaction with puberty, may be key factors contributing to learning problems.
Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage
Authors: Tang Qi-Qun , Otto Tamara C. , Lane M. Daniel
Published: June 2004

The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes. This hyperplasia results from recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. A model cell culture system has been developed that recapitulates this process both
ex vivo
and
in vivo
. After treatment of pluripotent C3H10T1/2 stem cells with bone morphogenic protein 4 (BMP4) during proliferation followed by differentiation inducers at growth arrest, the cells synchronously enter S phase and undergo mitotic clonal expansion, a hallmark of preadipocyte differentiation. Upon exiting the cell cycle, these cells express adipocyte markers and acquire adipocyte characteristics at high frequency. C3H10T1/2 cells treated with BMP4 in cell culture and implanted s.c. into athymic mice develop into tissue indistinguishable from adipose tissue in normal fat depots. We interpret the findings as evidence that BMP4 is capable of triggering commitment of pluripotent C3H10T1/2 stem cells to the adipocyte lineage.
Pulmonary delivery of an erythropoietin Fc fusion protein in non-human primates through an immunoglobulin transport pathway
Authors: Bitonti Alan J. , Dumont Jennifer A. , Low Susan C. , Peters Robert T. , Kropp Keith E. , Palombella Vito J. , Stattel James M. , Lu Yichun , Tan Cristina A. , Song Jeffrey J. , Garcia Ana Maria , Simister Neil E. , Spiekermann Gerburg M. , Lencer Wayne I. , Blumberg Richard S.
Published: June 2004

Administration of therapeutic proteins by methods other than injection is limited, in part, by inefficient penetration of epithelial barriers. Therefore, unique approaches to breaching these barriers are needed. The neonatal constant region fragment (Fc) receptor (FcRn), which is responsible for IgG transport across the intestinal epithelium in newborn rodents, is expressed in epithelial cells in adult humans and non-human primates. Here we show that FcRn-mediated transport is functional in the lung of non-human primates and that this transport system can be used to deliver erythropoietin (Epo) when it is conjugated to the Fc domain of IgG1. FcRn-dependent absorption was more efficient when the EpoFc fusion protein was deposited predominantly in the upper and central airways of the lung, where epithelial expression of FcRn was most prominently detected. To optimize fusion protein absorption in the lung, we created a recombinant “monomeric-Epo” Fc fusion protein comprised of a single molecule of Epo conjugated to a dimeric Fc. This fusion protein exhibited enhanced pharmacokinetic and pharmacodynamic properties. The bioavailability of the EpoFc monomer when delivered through the lung was approximately equal to that reported for unconjugated Epo delivered s.c. in humans. These studies show that FcRn can be harnessed to noninvasively deliver bioactive proteins into the systemic circulation in therapeutic quantities.
A-type lamins regulate retinoblastoma protein function by promoting subnuclear localization and preventing proteasomal degradation
Authors: Johnson Brett R. , Nitta Ryan T. , Frock Richard L. , Mounkes Leslie , Barbie David A. , Stewart Colin L. , Harlow Ed , Kennedy Brian K.
Published: June 2004

The retinoblastoma protein (pRB) is a critical regulator of cell proliferation and differentiation and an important tumor suppressor. In the G
1
phase of the cell cycle, pRB localizes to perinucleolar sites associated with lamin A/C intranuclear foci. Here, we examine pRB function in cells lacking lamin A/C, finding that pRB levels are dramatically decreased and that the remaining pRB is mislocalized. We demonstrate that A-type lamins protect pRB from proteasomal degradation. Both pRB levels and localization are restored upon reintroduction of lamin A.
Lmna
-/-
cells resemble
Rb
-/-
cells, exhibiting altered cell-cycle properties and reduced capacity to undergo cell-cycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cell-cycle regulator. They further raise the possibility that altered pRB function may be a contributing factor in dystrophic syndromes arising from
LMNA
mutation.
An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues
Authors: Liu Chang-Gong , Calin George Adrian , Meloon Brian , Gamliel Nir , Sevignani Cinzia , Ferracin Manuela , Dumitru Calin Dan , Shimizu Masayoshi , Zupo Simona , Dono Mariella , Alder Hansjuerg , Bullrich Florencia , Negrini Massimo , Croce Carlo M.
Published: June 2004

MicroRNAs (miRNAs) are a class of small noncoding RNA genes recently found to be abnormally expressed in several types of cancer. Here, we describe a recently developed methodology for miRNA gene expression profiling based on the development of a microchip containing oligonucleotides corresponding to 245 miRNAs from human and mouse genomes. We used these microarrays to obtain highly reproducible results that revealed tissue-specific miRNA expression signatures, data that were confirmed by assessment of expression by Northern blots, real-time RT-PCR, and literature search. The microchip oligolibrary can be expanded to include an increasing number of miRNAs discovered in various species and is useful for the analysis of normal and disease states.
Thermal-activated protein mobility and its correlation with catalysis in thermophilic alcohol dehydrogenase
Authors: Liang Zhao-Xun , Lee Thomas , Resing Katheryn A. , Ahn Natalie G. , Klinman Judith P.
Published: June 2004

Temperature-dependent hydrogen-deuterium (H/D) exchange of the thermophilic alcohol dehydrogenase (htADH) has been studied by using liquid chromatography-coupled mass spectrometry. Analysis of the changes in H/D exchange patterns for the protein-derived peptides suggests that some regions of htADH are in a rigid conformational substate at reduced temperatures with limited cooperative protein motion. The enzyme undergoes two discrete transitions at ≈30 and 45°C to attain a more dynamic conformational substate. Four of the five peptides exhibiting the transition above 40°C are in direct contact with the cofactor, and the NAD
+
-binding affinity is also altered in this temperature range, implicating a change in the mobility of the cofactor-binding domain >45°C. By contrast, the five peptides exhibiting the transition at 30°C reside in the substrate-binding domain. This transition coincides with a change in the activation energy of
k
cat
for hydride transfer, leading to a linear correlation between
k
cat
and the weighted average exchange rate constant
k
HX(WA)
for the five peptides. These observations indicate a direct coupling between hydride transfer and protein mobility in htADH, and that an increased mobility is at least partially responsible for the reduced
E
act
at high temperature. The data provide support for the hypothesis that protein dynamics play a key role in controlling hydrogen tunneling at enzyme active sites.
Tumor regionality in the mouse intestine reflects the mechanism of loss of
Apc
function
Authors: Haigis Kevin M. , Hoff Peter D. , White Alanna , Shoemaker Alex R. , Halberg Richard B. , Dove William F.
Published: June 2004

Inherited colorectal cancer syndromes in humans exhibit regional specificity for tumor formation. By using mice with germline mutations in the adenomatous polyposis coli gene (
Apc
) and/or DNA mismatch repair genes, we have analyzed the genetic control of tumor regionality in the mouse small intestine. In C57BL/6 mice heterozygous for the
Apc
multiple intestinal neoplasia mutation (
Apc
Min
), in which tumors are initiated by loss of heterozygosity by means of somatic recombination, tumors form preferentially in the distal region of the small intestine. By contrast, the formation of tumors initiated by allelic silencing on the AKR
Apc
Min
genetic background is strongly skewed toward the ileocecal junction. A third tumor regionality is displayed by tumors that develop in MMR-deficient
Apc
Min/+
mice, in which mutation of the
Apc
gene is responsible for tumor initiation. Thus, tumor regionality in the small intestine of
Apc
Min/+
reflects the mechanism by which the wild-type allele of
Apc
is inactivated. We have reexamined the mechanism of
Apc
loss in tumors from
Apc
1638N/+
mice, in which tumors of the small intestine develop in a regional pattern overlapping that of mismatch repair-deficient mice. In contrast to previous reports, we find that tumors from
Apc
1638N/+
mice on a congenic C57BL/6 background maintain the wild-type allele of
Apc
. Our studies demonstrate a pathway-specific regionality for tumor development in mouse models for inherited intestinal cancer, an observation that is reminiscent of the regional preference for tumor development in the human colon. Perhaps, the power of mouse genetics and biology can be harnessed to identify genetic and other factors that contribute to tumor regionality.
A fluorescence resonance energy transfer-derived structure of a quantum dot-protein bioconjugate nanoassembly
Authors: Medintz I. L. , Konnert J. H. , Clapp A. R. , Stanish I. , Twigg M. E. , Mattoussi H. , Mauro J. M. , Deschamps J. R.
Published: June 2004

The first generation of luminescent semiconductor quantum dot (QD)-based hybrid inorganic biomaterials and sensors is now being developed. It is crucial to understand how bioreceptors, especially proteins, interact with these inorganic nanomaterials. As a model system for study, we use Rhodamine red-labeled engineered variants of
Escherichia coli
maltose-binding protein (MBP) coordinated to the surface of 555-nm emitting CdSe-ZnS core–shell QDs. Fluorescence resonance energy transfer studies were performed to determine the distance from each of six unique MBP-Rhodamine red dye-acceptor locations to the center of the energy-donating QD. In a strategy analogous to a nanoscale global positioning system determination, we use the intraassembly distances determined from the fluorescence resonance energy transfer measurements, the MBP crystallographic coordinates, and a least-squares approach to determine the orientation of the MBP relative to the QD surface. Results indicate that MBP has a preferred orientation on the QD surface. The refined model is in agreement with other evidence, which indicates coordination of the protein to the QD occurs by means of its C-terminal pentahistidine tail, and the size of the QD estimated from the model is in good agreement with physical measurements of QD size. The approach detailed here may be useful in determining the orientation of proteins in other hybrid protein–nanoparticle materials. To our knowledge, this is the first structural model of a hybrid luminescent QD-protein receptor assembly elucidated by using spectroscopic measurements in conjunction with crystallographic and other data.
T-cadherin is a receptor for hexameric and high-molecular-weight forms of Acrp30/adiponectin
Authors: Hug Christopher , Wang Jin , Ahmad Naina Shehzeen , Bogan Jonathan S. , Tsao Tsu-Shuen , Lodish Harvey F.
Published: June 2004

Acrp30/adiponectin is reduced in the serum of obese and diabetic individuals, and the genetic locus of adiponectin is linked to the metabolic syndrome. Recombinant adiponectin, administered to diet-induced obese mice, induced weight loss and improved insulin sensitivity. In muscle and liver, adiponectin stimulates AMP-activated protein kinase activation and fatty acid oxidation. To expression-clone molecules capable of binding adiponectin, we transduced a C2C12 myoblast cDNA retroviral expression library into Ba/F3 cells and panned infected cells on recombinant adiponectin linked to magnetic beads. We identified T-cadherin as a receptor for the hexameric and high-molecular-weight species of adiponectin but not for the trimeric or globular species. Only eukaryotically expressed adiponectin bound to T-cadherin, implying that posttranslational modifications of adiponectin are critical for binding. An adiponectin mutant lacking a conserved N-terminal cysteine residue required for formation of hexamer and high-molecular-weight species did not bind T-cadherin in coimmunoprecipitation studies. Although lacking known cellular functions, T-cadherin is expressed in endothelial and smooth muscle cells, where it is positioned to interact with adiponectin. Because T-cadherin is a glycosylphosphatidylinositol-anchored extracellular protein, it may act as a coreceptor for an as-yet-unidentified signaling receptor through which adiponectin transmits metabolic signals.
Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity
Authors: Buchholz Ursula J. , Bukreyev Alexander , Yang Lijuan , Lamirande Elaine W. , Murphy Brian R. , Subbarao Kanta , Collins Peter L.
Published: June 2004

We investigated the contributions of the structural proteins of severe acute respiratory syndrome (SARS) coronavirus (CoV) to protective immunity by expressing them individually and in combinations from a recombinant parainfluenza virus (PIV) type 3 vector called BHPIV3. This vector provided direct immunization of the respiratory tract, the major site of SARS transmission, replication, and disease. The BHPIV3/SARS recombinants were evaluated for immunogenicity and protective efficacy in hamsters, which support a high level of pulmonary SARS-CoV replication. A single intranasal administration of BHPIV3 expressing the SARS-CoV spike protein (S) induced a high titer of SARS-CoV-neutralizing serum antibodies, only 2-fold less than that induced by SARS-CoV infection. The expression of S with the two other putative virion envelope proteins, the matrix M and small envelope E proteins, did not augment the neutralizing antibody response. In absence of S, expression of M and E or the nucleocapsid protein N did not induce a detectable serum SARS-CoV-neutralizing antibody response. Immunization with BHPIV3 expressing S provided complete protection against SARS-CoV challenge in the lower respiratory tract and partial protection in the upper respiratory tract. This was augmented slightly by coexpression with M and E. Expression of M, E, or N in the absence of S did not confer detectable protection. These results identify S among the structural proteins as the only significant SARS-CoV neutralization antigen and protective antigen and show that a single mucosal immunization is highly protective in an experimental animal that supports efficient replication of SARS-CoV.
G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment
Authors: Atreya Hanudatta S. , Szyperski Thomas
Published: June 2004

A G-matrix Fourier transform (GFT) NMR spectroscopy-based strategy for resonance assignment of proteins is described. Each of the GFT NMR experiments presented here rapidly affords four-, five-, or six-dimensional spectral information in combination with precise measurements of chemical shifts. The resulting high information content enables one to obtain nearly complete assignments by using only four NMR experiments. For the backbone amide proton detected “out-and-back” experiments, data collection was further accelerated up to ≈2.5-fold by use of longitudinal
1
H relaxation optimization. The GFT NMR experiments were acquired for three proteins with molecular masses ranging from 8.6 to 17 kDa, demonstrating that the proposed strategy is of key interest for automated resonance assignment in structural genomics.
Hypoxic stress tolerance of the blind subterranean mole rat: Expression of erythropoietin and hypoxia-inducible factor 1α
Authors: Shams Imad , Avivi Aaron , Nevo Eviatar
Published: June 2004

Blind subterranean mole rats (Spalax, Spalacidae) evolved adaptive strategies to cope with hypoxia that climaxes during winter floods in their burrows. By using real-time PCR, we compared gene expression oferythropoietin(Epo), a key regulator of circulating erythrocytes, andhypoxia-inducible factor 1α (HIF-1α),Epoexpression inducer, in the kidneys ofSpalaxand white rats,Rattus norvegicus. Our results show significantly higher, quicker, and longer responses to different O2levels inSpalaxcompared withRattus. (i) In normoxia, bothSpalaxandRattuskidneys produce small amounts ofEpo. Maximal expression ofRattus Epois noticed after a 4-h hypoxia at 6% O2. Under these conditions,Spalax Epolevels are 3-fold higher than inRattus. After 24 h of 10% O2,Spalax Eporeaches its maximal expression, remarkably 6-fold higher than the maximum inRattus; (ii) theHIF-1α level in normoxia is 2-fold higher inSpalaxthan inRattus. Spalax HIF-1α achieves maximal expression after 4-h hypoxia at 3% O2, a 2-fold increase compared with normoxia, whereas no significant change was detected inRattus HIF-1α at any of the conditions studied; (iii) at 6% O2for 10 h, in whichRattuscannot survive,EpoandHIF-1α levels inSpalax galili, living in heavily flooded soils, are higher than inSpalax judaei, residing in light aerated soil. We suggest that this pattern ofEpoandHIF-1α expression is a substantial contribution to the adaptive strategy of hypoxia tolerance inSpalax, evolved during 40 million years of evolution to cope with underground hypoxic stress.
Poly-β amino ester-containing microparticles enhance the activity of nonviral genetic vaccines
Authors: Little Steven R. , Lynn David M. , Ge Qing , Anderson Daniel G. , Puram Sidharth V. , Chen Jianzhu , Eisen Herman N. , Langer Robert
Published: June 2004

Current nonviral genetic vaccine systems are less effective than viral vaccines, particularly in cancer systems where epitopes can be weakly immunogenic and antigen-presenting cell processing and presentation to T cells is down-regulated. A promising nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect plasmid payloads and target them to phagocytic antigen-presenting cells. However, conventional microparticle formulations composed of poly lactic-co-glycolic acid take too long to release encapsulated payload and fail to induce high levels of target gene expression. Here, we describe a microparticle-based DNA delivery system composed of a degradable, pH-sensitive poly-β amino ester and poly lactic-co-glycolic acid. These formulations generate an increase of 3–5 orders of magnitude in transfection efficiency and are potent activators of dendritic cellsin vitro. When used as vaccinesin vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells.
Apolipoprotein CIII promotes Ca
2+
-dependent β cell death in type 1 diabetes
Authors: Juntti-Berggren Lisa , Refai Essam , Appelskog Ioulia , Andersson Mats , Imreh Gabriela , Dekki Nancy , Uhles Sabine , Yu Lina , Griffiths William J. , Zaitsev Sergei , Leibiger Ingo , Yang Shao-Nian , Olivecrona Gunilla , Jörnvall Hans , Berggren Per-Olof
Published: June 2004

In type 1 diabetes (T1D), there is a specific destruction of the insulin secreting pancreatic β cell. Although the exact molecular mechanisms underlying β cell destruction are not known, sera from T1D patients have been shown to promote Ca
2+
-induced apoptosis. We now demonstrate that apolipoprotein CIII (apoCIII) is increased in serum from T1D patients and that this serum factor both induces increased cytoplasmic free intracellular Ca
2+
concentration ([Ca
2+
]
i
) and β cell death. The apoCIII-induced increase in [Ca
2+
]
i
reflects an activation of the voltage-gated L-type Ca
2+
channel. Both the effects of T1D sera and apoCIII on the β cell are abolished in the presence of antibody against apoCIII. Increased serum levels of apoCIII can thus account for the increase in β cell [Ca
2+
]
i
and thereby β cell apoptosis associated with T1D.
Direct evidence that a conserved arginine in RuvB AAA
+
ATPase acts as an allosteric effector for the ATPase activity of the adjacent subunit in a hexamer
Authors: Hishida Takashi , Han Yong-Woon , Fujimoto Satoko , Iwasaki Hiroshi , Shinagawa Hideo
Published: June 2004

The
Escherichia coli
RuvA and RuvB protein complex promotes branch migration of Holliday junctions during recombinational repair and homologous recombination and at stalled replication forks. The RuvB protein belongs to the AAA
+
(ATPase associated with various cellular activities) ATPase family and forms a hexameric ring in an ATP-dependent manner. Studies on the oligomeric AAA
+
class ATPases suggest that a conserved arginine residue is located in close proximity to the ATPase site of the adjacent subunit and plays an essential role during ATP hydrolysis. This study presents direct evidence that Arg-174 of RuvB allosterically stimulates the ATPase of the adjacent subunit in a RuvB hexamer. RuvBR174A shows a dominant negative phenotype for DNA repair
in vivo
and inhibits the branch migration catalyzed by wild-type RuvB. A dominant negative phenotype was also observed with RuvBK68A (Walker A mutation). RuvB K68A–R174A double mutant demonstrates a more severe dominant negative effect than the single mutants RuvB K68A or R174A. Moreover, although RuvB K68A and R174A are totally defective in ATPase activity, ATPase activity is restored when these two mutant proteins are mixed at a 1:1 ratio. These results suggest that each of the two mutants has distinct functional defects and that restoration of the ATPase activity is brought by complementary interaction between the mutant subunits in the heterohexamers. This study demonstrates that R174 plays an intermolecular catalytic role during ATP hydrolysis by RuvB. This role may be a general feature of the oligomeric AAA/AAA
+
ATPases.
Calcium, with magnesium, is essential for normal seedling development from partially dehydrated recalcitrant axes: a study on Trichilia dregeana Sond.
Authors: Berjak Patricia , Mycock David
Published: June 2004

Assessment of the rehydration procedure has been consistently overlooked in evaluation of factors contributing to successful cryostorage of partially dehydrated, embryonic axes excised from recalcitrant seeds. Conventional rehydration of Trichilia dregeana (Sond.) axes in distilled water resulted in the lack of root pole gravitropism after culture on medium in vitro. In comparison, a strong gravitropic response was observed in axes that had not been dehydrated, and by those rehydrated in a solution containing 1 M CaCl2 and 1 mM MgCl2. However, no marked loss of either cation from the tissue could be detected, whether axes were rehydrated in distilled water or the Ca2+/Mg2+ solution. Starch-packed statoliths differentiated rapidly in both non-dehydrated axes and those rehydrated in the divalent cation solution, but these organelles failed to develop or accumulate much starch following axis rehydration in water, as was the case for the amyloplasts of root cells generally. After rehydration in the Ca2+/Mg2+ solution and 48 h in culture, axis root-cap columella cells accumulated Ca2+, whereas axes rehydrated in water or solutions containing either Ca2+ or Mg2+ alone did not take up Ca2+. Rehydration with the Ca2+/Mg2+ solution also altered distribution of the actin component of the cytoskeleton. When rehydrated in the divalent cation solution, actin was associated with the nucleus and with the statoliths, which were located distally in statocytes of axes. In contrast, actin was largely confined to the perinuclear area in root-cap columella cells of the agravitropic, water-rehydrated axes. The present results indicate a definitive primary role for Ca2+ with Mg2+ in graviperception, via starch metabolism, and in the determination of statolith morphology, which appears to be linked with maintenance of the actin component of the cytoskeleton in root-cap statocytes.
Developmental status is a critical factor in the selection of excised recalcitrant axes as explants for cryopreservation: a study on Trichilia dregeana Sond.
Authors: Goveia Meagan , Kioko Joseph I. , Berjak Patricia
Published: June 2004

As a consequence of previous lack of success in cryostoring axes excised from newly shed seeds of Trichilia dregeana, the effects of the mode of axis excision on seedling production were investigated. Although vigorous root production occurred, no shoots were produced when the cotyledons were severed as closely as possible to the axis surface (explant-type 0). In contrast, shoot production was increasingly facilitated when small to larger segments of cotyledonary tissue were left attached to the axes (explant-types 1, 2 and 4), which did not compromise axis drying rate. However, root growth of explant-types 1, 2 and 4 was negatively affected, probably by leakage into the medium of an inhibitory or toxic substance(s) from the cut surfaces of the cotyledonary tissue. Microscopical examination revealed that the cotyledons were sessile, and their insertions were contiguous with the shoot apex in axes from newly shed seeds, leading to the suggestion that failure of shoot production by type 0 explants in vitro was the direct consequence of the proximity of the wound sites to the apical meristem. When seeds were stored hydrated for 6 months, the shoot apex had elongated, positioning the apical meristem some distance from the top of the cotyledonary insertions. In contrast to axes excised as type 0 explants from newly shed seeds, the equivalent explants from the stored seeds rapidly formed shoots and leaves in vitro. This indicates that the developmental status of axes, when excised, dictates failure or success in their further development in vitro, and that this aspect needs to be resolved before any further manipulations for cryostorage are attempted.
Water potential of cherry tomato (Lycopersicon esculentum Mill.) placenta and seed germination in response to desiccation during fruit development
Authors: Modi Albert T. , White Belinda J.
Published: June 2004

Cherry tomato (Lycopersicon esculentumMill.) seeds harvested from fruits at four stages of development [2, 4, 6 and 8 weeks after flowering (WAF)] were washed (1% HCl) and germinated after 72 h of desiccation at 20°C, and 76%, 49% or 12% RH. Seed α-amylase activity was determined at each stage of development and correlated with seed germination. Desiccation at 76% and 49% RH had no significant effect on the germination of seeds at 4, 6 and 8 WAF, whereas it improved germination of seeds at 2 WAF. Low RH (12%) significantly reduced seed germination at all stages of development. There was a positive correlation (r= 0.68,P≤ 0.05) between germination and α-amylase activity during seed development. Scanning electron microscopy also revealed a correlation between starch grain occurrence in the endosperm and α-amylase activity. Placenta water potential decreased with fruit and seed maturation, and correlated negatively with improved seed germination and α-amylase activity. Promotion of seed germination by desiccation in developing cherry tomato seeds was lost 4 weeks prior to mass maturity (80 ± 3% seed water content and –2.3 ± 0.1 MPa placenta water potential). It is proposed that the water potential differential between the placenta and the seeds influences α-amylase activity and germination behaviour during development.
FILTRATION AND REABSORPTION OF PROTEIN BY THE KIDNEY
Authors: Sellers Alvin L. , Griggs Neilyn , Marmorston Jessie , Goodman Howard C.
Published: June 2004

Plasma proteins of the rat have been labelled by the in vivo injection of the dye T-1824.

From a study of the rate of disappearance of T-1824 from the circulating blood, and the total T-1824 content of the perfused kidney the rate of protein reabsorption from the glomerular fluid by the cells of the renal tubule has been calculated.

It is concluded that protein reabsorption by the cells lining the proximal convoluted tubule of the rat kidney proceeds at a rate of at least 5 mg. per hour, equivalent to a daily filtration and reabsorption of 33 per cent of the circulating plasma protein.
THE BINDING OF PENICILLIN IN RELATION TO ITS CYTOTOXIC ACTION
Authors: Eagle Harry
Published: June 2004

1. In a previous study, the differing sensitivity of bacterial strains as they occur in nature appeared to be correlated with their correspondingly differing reactivity with penicillin. Presumably, the over-all reactivity of the cell with penicillin paralleled that of the vulnerable cell component(s). However, when penicillin-resistant variants of these strains (Streptococcus pyogenes, Micrococcus pyogenes, Diplococcus pneumoniae, and Streptococcus faecalis) were produced by serial passage through increasing concentrations of antibiotic, this correlation between resistance and the ability of the cell to bind penicillin was no longer apparent. Some resistant variants bound more penicillin than the parent, sensitive cell (Streptococcus faecalis, Micrococcus pyogenes), some were unchanged in their reactivity (Diplococcus pneumoniae, Micrococcus pyogenes), and some bound less (Streptococcus pyogenes, Micrococcus pyogenes). One resistant variant of Micrococcus pyogenes at first showed enhanced reactivity with penicillin; on continued passage through antibiotic, there was a further increase in resistance, but now associated with a significantly decreased reactivity. In the case of Diplococcus pneumoniae, a resistant variant at first reacted normally with penicillin; on continued passage in antibiotic, its binding affinity for penicillin gradually decreased, but with no associated further increase in resistance.

2. The reactivity with penicillin of cell-free sonic extracts of the resistant variants paralleled that of the intact organisms. Permeability considerations therefore did not seem involved in the increased resistance produced by serial passage in antibiotic.

3. The penicillin-resistant variants did not have an enhanced capacity to degrade the free intracellular antibiotic.

4. Possible alternative explanations are discussed in the text.
VITAMIN D AND ENDOCHONDRAL OSSIFICATION IN THE RAT AS INDICATED BY THE USE OF SULFUR-35 AND PHOSPHORUS-32
Authors: Dziewiatkowski Dominic D.
Published: June 2004

The concentration of inorganic sulfate-sulfur in the serum of vitamin D-deficient rats, 2.6 to 3.5 mg. per cent, was found to be higher than that in the serum of normal rats of the same age, 2.0 mg. per cent. No change was observed following the administration of 25 γ of vitamin D2.

In accord with the results of others, it was found that a definitely increased deposition of phosphorus in femurs and tibiae had occurred 36 to 48 hours after the administration of vitamin D2 to vitamin D-deficient rats. An immediate increase in the uptake of sulfate by the skeleton was found using sodium sulfate-S35.

As measured by the specific activity of sulfate-sulfur in samples of chondroitin sulfate isolated from the skeletons of the vitamin D-deficient animals and from normal controls receiving equal doses of sulfur-35, the rate of synthesis of chondroitin sulfate in rachitic rats is similar to the rate in normal rats of the same age. Likewise, the incorporation of labelled sulfate into the sulfomuco-polysaccharides of the pelts was found to be equal at 12 hours to that in normal rats. Following the administration of vitamin D2 to deficient animals an increase in the rate of synthesis of the chondroitin sulfate of the skeletons was noted.

The radiochemical and radioautographic evidence suggest that there is in vitamin D-deficient rats an impaired utilization of chondroitin sulfate and that vitamin D2 is able to accelerate this process.
STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM
Authors: Finter Norman B. , Liu Oscar C. , Lieberman Melvin , Henle Werner
Published: June 2004

The usefulness of the deembryonation technic has been analyzed as a tool in the study of various problems in the growth cycle of influenza virus in the entodermal cells of the allantoic of chick embryos.

Various improvements in the deembryonation technic have been described.

The method readily permits repeated sampling of the medium at various stages after infection (cumulative growth curves) or frequent exchanges of the medium (differential growth curve). However, the yield of infectious virus or of hemagglutinins is less than that observed in the intact chick embryo. The difference observed is greater than can be accounted for by the reduction in the available host cells and is assumed, therefore, to be due in part to interruption of blood and nutrient supply to the cells. This handicap can be overcome by the combined in ovo-deembryonation technic, in which deembryonation is performed at any desired time after infection of the intact chick embryo, and the medium is collected and analyzed after 1 to 3 hours of further incubation.

The value of the technic is demonstrated by the fact that liberation of virus from infected cells can be detected earlier than in the intact egg. Furthermore, it continues at a nearly constant rate for many hours, thus proving to be erroneous previous inference which had been based upon in ovo experiments.

The technic also permits readily the addition and subsequent removal of substances that might interfere with viral propagation. As an example a study was made of the effect of the receptor-destroying enzyme of V. cholerae (RDE) when added to the medium of eggs infected prior to deembryonation. By carefully grading the dose of virus and using an appropriate amount of RDE, one-step growth curves were obtained indicating that those cells not directly invaded by the seed virus were subsequently protected against infection by action of the enzyme. The smaller the amount of virus the less RDE was required in order to note a protective effect. With a decrease in the period of exposure to RDE regeneration of cell receptors became increasingly more apparent in that correspondingly greater amounts of virus were produced and liberated late in the incubation periods. These results confirmed and extended those reported by Stone.

More extensive applications of these technics will be reported in subsequent papers of this series.
THE OCCURRENCE DURING ACUTE INFECTIONS OF A PROTEIN NOT NORMALLY PRESENT IN THE BLOOD
Authors: Wood Harrison F. , McCarty Maclyn , Slater Robert J.
Published: June 2004

A method is described for obtaining crystalline C-reactive protein from serous fluids in which the protein is associated with lipid. Most pathological fluids currently available as a source of this protein appear to fall in this category.

Crystalline C-reactive protein has its isoelectric point at pH 4.82 as determined by free electrophoresis in McIlvaine's buffer. Its mobility in the electrophoresis cell, both alone and after addition to normal serum, coincides with that of the ß-globulin fraction of the serum. In contrast to this finding, by the method of zone electrophoresis on a starch supporting medium the protein migrates with the γ1-globulin. The significance of this discrepancy is discussed. Studies in the ultracentrifuge indicate an s20,w of 7.5.
THE EFFECTS ON BIOLOGICAL MATERIALS OF FREEZING AND DRYING BY VACUUM SUBLIMATION
Authors: Greiff Donald , Pinkerton Henry
Published: June 2004

A vacuum sublimation apparatus is described which will permit, (a) the removal of water from virus suspensions at temperatures ranging down to –80°C., (b) continuous operation with a minimum of attention from the investigator, (c) sealing off of samples at operating pressures (10–5 mm. Hg), (d) simultaneous lyophilization of aliquot samples at different temperatures, (e) isolation of a portion of the apparatus without disturbing the remainder of the system, and (f) determination of the end-point of sublimation without disturbing the samples.

The time required for drying 0.1 ml. of influenza virus suspension was shown to increase markedly with decrease of temperature, 8 days being required for dehydration at –80°C. in contrast to 2 days at –30°C. and 1 day at 0°C.
THE EFFECTS ON BIOLOGICAL MATERIALS OF FREEZING AND DRYING BY VACUUM SUBLIMATION
Authors: Greiff Donald , Blumenthal Herman , Chiga Masahiro , Pinkerton Henry
Published: June 2004

The infectivity titre of influenza virus-infected allantoic fluid was determined after a variety of procedures involving cyclic slow freezing and thawing, freezing at various rates with subsequent storage at different temperatures freezing at various rates with subsequent dehydration at various temperatures, and different degrees of dehydration. All these factors were found to influence the survival rate of the virus particles.

Five freeze-thaw cycles resulted in a fall in titre from 10–8.6 to 10–0.8 cycles 2, 3, and 4 causing much greater losses than cycles 1 and 5. Rapid cooling to –40°C. or slow cooling to –80 or 190°C. did not cause significant titre loss, but rapid cooling to temperatures above –40° or slow cooling to temperatures above –80°C. caused definite titre loss. Loss of titre on storage occurred only at temperatures above –40deg;C.

The effect of lyophilization depends both on the preliminary treatment and on the dehydration temperature. Better conservation of titre was obtained after preliminary cooling to –190 or –80°C. than after preliminary cooling to higher temperatures. The most effective sublimation temperatures were 0 and –80°.; the least effective was +20°C. Titre losses in suspensions sublimated at –10, –30, and –60°C. were in general intermediate. No loss in titre occurred after preliminary cooling to –80 or –190°C. and subsequent dehydration at –80 or 0°C. The degree of dehydration definitely affects the survival of virus on storage at 0°C., but sublimation for 4 hours at 0°C. gave complete protection against titre loss on storage at this temperature.

Possible explanations of the observations made are suggested, based on known physiochemical phenomena such as supercooling, vitrification, variations in size and shape of ice crystals with different freezing speeds, differential enzyme inactivation, changes in salt concentration, and changes in energy levels.
SIGNIFICANCE OF THE HYDROXYL GROUPS OF STEROIDS IN PROMOTING GROWTH
Authors: Huggins Charles , Jensen Elwood V.
Published: June 2004

The presence of a 17ß-hydroxyl group endows the simple androstane molecule with the ability to produce growth of the uterus, vagina, and prostate of the female hypophysectomized albino rat. It appears that hydrogen atoms at position 17 are of critical importance since related compounds with a ketone group at this site are inactive. Monofunctional steroids with a hydroxyl or a ketone group at position 3 likewise are devoid of activity.

If a phenolic A-ring is present in monofunctional steroids the 17ß-hydroxyl group is not obligatory for growth. Proliferation of the uterus and vagina were found to follow the administration of 17-desoxyestradiol.
STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE
Authors: Morgan Councilman , Ellison Solon A. , Rose Harry M. , Moore Dan H.
Published: June 2004

Vaccinia and fowl pox viruses were visualized by the electron microscope in sections of infected chorioallantoic membrane of chicken embryos. The viruses were of similar structure and size, averaging 200 x 300 mµ with considerable individual variation. Intracytoplasmic viral particles contained a dense, nucleus-like body (nucleoid) separated from granular material (viroplasm) by a zone of lesser density. They were enclosed by a single membrane. Near the surface of the host cell and in the extracellular space the particles consisted of a central body of variable shape and density enclosed by a double membrane. The initial sites of development were confined to the cytoplasm of the host cell. Before release from the host cell the viral nucleoids appeared to enlarge and to occupy a central position within the particle, which became enclosed by a double limiting membrane. The brick-shaped forms found after removal of the embedding plastic from thick sections indicated that drying caused characteristic distortion of certain viral particles.
STUDIES ON ENTRY AND EGRESS OF POLIOMYELITIC INFECTION
Authors: Faber Harold K. , Dong Luther
Published: June 2004

At 56, 60, and 72 hours after simple feeding of poliomyelitis virus, typical, discrete lesions were found in the ganglia supplying the mouth and pharynx, which were most numerous and severe in the Gasserian ganglia. Lesions were also found in the nerve bundles adjacent to the infected ganglia.

The character, localizations, and time of appearance of lesions point to nerve-conducted entry of infection from the mucosa of the mouth and pharynx.

The possibility is suggested that under natural conditions of exposure, in which only small amounts of virus are involved, artificially induced immunity, active and probably passive, may block primary neural entry at the oropharyngeal portal by virtue of antibodies in the overlying mucus.
IMMUNOCHEMICAL STUDY OF A BACTERIAL ENDOTOXIN: SHIGELLA FLEXNERI TYPE Z
Authors: Cluff Leighton E.
Published: June 2004

The endotoxins of the Gram-negative bacteria have similar biological and chemical properties. The toxic antigen of Shigella flexneri Type Z was selected as a representative endotoxin, and it was confirmed that the antigen consists of a polysaccharide conjugated with phospholipid and protein. By the technique of zone electrophoresis, the polysaccharide of the purified endotoxin was shown to be conjugated with each of three different proteins, and each conjugate proved toxic and antigenic for the rabbit. Two of the protein conjugates were digested by trypsin and the released polysaccharide appeared to conjugate with the remaining trypsin-resistant protein. Immunological analysis revealed that the purified toxic antigen is heterogeneous and that the polysaccharide and protein components possessed serological activity. The trypsin-treated toxin had a single electrophoretic zone and its precipitation in gel suggested immunological purity. Proteolytic treatment of the endotoxin did not destroy its toxicity or antigenicity for the rabbit.
OSMOTIC ACTIVITY OF TISSUES DURING FETAL AND POSTNATAL GROWTH
Authors: Opie Eugene L.
Published: June 2004

The osmotic pressure maintained by liver tissue of the white rat preceding birth is less than that of the maternal blood serum and shortly after birth approximates this level.

Following birth osmotic pressure of liver tissue, continuing to increase, reaches after about 60 to 90 days the level found in the liver of mature animals and is then isotonic with solutions of sodium chloride with concentration slightly more than twice that isotonic with blood serum.

Osmotic pressure maintained by kidney tissue pursues with growth a similar course but at a lower level and about 35 to 60 days after birth reaches that found in the mature animal being represented by isotonicity with a concentration of sodium chloride slightly less than twice that isotonic with blood serum.

The tissues of the whole fetus are isotonic with sodium chloride solutions less concentrated than that isotonic with the maternal blood serum.