SELECTIVE RETENTION AND FILTRATION OF BRAIN NUCLEIC ACIDS IN AGAROSE GELS

Abstract— Total nucleic acids of rat brain have been separated by agarose gel chromatography at 2 m -NaCl into DNA. transfer RNA plus low molecular weight RNA. and high molecular weight RNA fractions. The DNA fraction contained less than 1 per cent RNA by weight judged by either short-term or long-term labelling with ortho[³²P]phosphate. The high molecular weight RNA fraction contained 28 s and 18 s ribosomal RNAs and a heterogeneous population of 20-60 s RNAs, apparent after short-term labelling and characterized by a high content of nearest-neighbour-labelled uridylic acid. The rapidly sedimenting (>30 s ) portion of these RNAs could be largely separated from ribosomal RNAs by gel filtration using 4% agarose. The ribosomal RNAs could be fully resolved into 28 s and 18 s components by agarose gel chromatography at 0.5 m -0.6 m -NaCl, as shown by analysis of their sedimentation and nucleotide composition.     

Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells

AIM: It was the aim of this study to prepare purified DNA/PEI polyplexes, which are coated with hyaluronan to facilitate CD44 receptor mediated uptake of the DNA/PEI polyplex and to reduce unspecific interactions of the complex with negatively charged extracellular matrix components on the ocular surface. METHODS: Hyaluronans of different molecular weights (<10 kDa, 10-30 kDa and 30-50 kDa) were isolated after enzymatic degradation of high molecular weight hyaluronan via ultrafiltration by centrifugation. The influence of the different hyaluronans used for coating on the stability and transfection efficiency of the complexes was evaluated in vitro. Transfection and uptake studies were performed in human corneal epithelial (HCE) cells. CD44 receptor expression of this cell model was evaluated by immunohistochemistry. RESULTS: Coating of purified DNA/PEI polyplexes with low molecular weight hyaluronan (<10 kDa) facilitated receptor-mediated uptake via the CD44 receptor in HCE cells, increased complex stability in vitro, and effectively shielded the positive surface charges of the polyplex without decreasing its transfection efficiency. Higher molecular weights and larger amounts of hyaluronan in the complexes resulted in lesser improvements in the stability and transfection efficacy of the complexes. CONCLUSIONS: Coating of polyplexes with low molecular weight hyaluronan is a promising strategy for gene delivery to the ocular surface, where CD44 receptor mediated uptake decreased cytotoxicity and reduced non-specific interactions with the negatively charged extracellular matrix components are considered beneficial for increased transfection efficiency of non-viral vectors.     

Statistical optimization of medium components for extracellular protease production by an extreme haloarchaeon, Halobacterium sp. SP1(1)

Aims:  Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach.
Methods and Results:  The significant factors influencing the protease production as screened by Plackett–Burman method were identified as soybean flour and FeCl₃. Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l⁻¹): NaCl, 250; KCl, 2; MgSO₄, 10; tri-Na-citrate, 1·5; soybean flour, 10 and FeCl₃, 0·16. This statistical optimization approach led to production of 69·44 ± 0·811 U ml⁻¹ of protease.
Conclusions:  Soybean flour and FeCl₃ were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3·9-fold increase in extracellular protease production.
Significance and Impact of the Study:  The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material.     

Characterization of intra- and extracellular transforming growth factors from normal and avian sarcoma virus-transformed rat cells

Intra- and extracellular transforming growth factors (TGFs) have been characterized in an avian sarcoma virus-transformed rat cell line, 77N1, and the nontransformed parental cell line, NRK. Serum-free conditioned medium from 77N1 and cell extracts from NRK and 77N1 were subjected to ion exchange column chromatography. Intracellular TGFs in cell extracts of NRK and 77N1 cells showed a major peak of DNA synthesis-stimulating and colony-forming activities which eluted in the 0.10 to 0.15 M salt concentration region. Extracellular TGF in conditioned medium of 77N1 cells showed a major peak of activity which eluted at 0.05 to 0.06 M salt concentration. Furthermore partially purified extracellular TGF had a molecular weight of about 40,000 daltons, whereas that for intracellular TGF was about 12,000 daltons. These intra- and extracellular TGFs, as well as TGF gamma 2 which was purified from 77N1 cell extract, were heat- and acid-labile polypeptides sensitive to treatment with dithiothreitol. Radioimmunoprecipitation analyses with antiserum against TGF gamma 2 demonstrated that intra- and extracellular TGFs in 77N1 and NRK cells were immunologically identical or very closely related to each other and suggested the possibility that extracellular TGF from 77N1 cells was released into serum-free conditioned medium after formation of complex with other cellular components.     

Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions.     

Biochemical properties of fat body DNA of the silkworm, Bombyx mori

Some properties of DNA, especially of pupal fat body of the silkworm, Bombyx mori, were studied. Pupal fat body DNA was separated into at least three components called -DNA, β₁-DNA, and β₂-DNA on methylated albumin kieselguhr (MAK) column chromatography. All of these classes of DNA were demonstrated to be pure DNA, neither contaminated nor hybridized with RNA, by their being positive to the diphenylamine reaction, sensitive to DNase, resistant to RNase, and incorporating thymidine-6-³H but not uridine-5-³H. The GC contents calculated from T_m values were around 38 per cent for all of these three components, almost coinciding with that of bulk DNA. But the molecular weight of -DNA, roughly calculated from the sedimentation coefficient on a sucrose density gradient centrifugation was several-fold larger than that of β₁-DNA.

In the pupal stage, fat body DNA was mainly composed of β₁- and β₂-DNA with a minor amount of -DNA, while in larval stage, it consisted only of -DNA. Larval fat body type DNA was observed in the larval silk gland, and in pupal and/or pharate adult tissues like the integument, muscles, and gonads. On the other hand, pupal fat body type DNA was detected in the tissues destined to degenerate or in the process of degeneration, such as pupal silk gland and midgut. These facts indicate that β₁- and β₂-DNA may be the degradation products of -DNA.     

Phosphorus transformations in the epilimnion of humic lakes: abiotic interactions between dissolved humic materials and phosphate

SUMMARY. 1. Sephadex gel filtration of filtered water from small, Finnish forest lakes demonstrated abiotic movement of ³³P from added PO₄ to two higher molecular weight fractions. This movement was most pronounced in waters with high humic content which also had high iron content. The two fractions which took up ¹³P had nominal molecular weights of > 100,000 and 10,000-20,000.
2. An equilibrium existed between free PO₄ and the two fractions. However, one fraction, at least, appeared to exist in two phases, with one phase in rapid equilibrium with free PO₄ but the other in only slow equilibrium.
3. Additions of ferric iron up to 1 mg Fe l⁻¹ to the filtered lake water stimulated movement from free PO₄, provided high concentrations of humic materials were present. In the absence of humic materials even 0.1 mg Fe 1⁻¹ would precipitate all added ³³PO₄.
4. The high molecular weight P was only partially reactive with standard molybdate reagents. Exposure of the high molecular weight P to sunlight caused a small release of PO₄ under the experimental conditions employed.
5. Possible implications for biological phosphorus demand of such sequestration of free PO₄ by humic materials in combination with iron are discussed.     

The Euglena gracilis chloroplast genome: analysis by restriction enzymes.

1. Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells. 2. Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4-7 X 10(6) and G+C-rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients. 3. Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G+C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R-EcoRI, end R-HindII, endoR-HindIII, endo R-HindII+III, endoR-Hpal, endo R-HpaII and endoR-HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions. 4. With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions. 5. Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns. 6. Digestion of total DNA with the endoR-HaeIII yielded 51-52 fragments separated in the gels in a total of 36 bands of which 11-12 bands were composed of 2-3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 X 10(6) or 95% of the genome (92 X 10(6)). 7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr92 X 10(6)). These cistrons are located on seven different types of endo R-HaeIII fragments. The hybridising fragments are preferentially found in the G+C-rich subfraction and in bands which are composed of 2-3 fragments.     

Geochemical aspects of aqueous iron, phosphorus and dissolved organic carbon in the humic Lake Tjeukemeer, The Netherlands

SUMMARY. 1. The main source of P, Fe and DOC in the humic Lake Tjeukemeer is superfluous water pumped from surrounding peaty polders. Most particulate P is intracellular but almost all particulate Fe is abiotic.
2. The size and molecular weight of the P, Fe and DOC (mainly fulvic acids (FA)) were determined by ultrafiltration and Sephadex G-100 gel filtration. Throughout the year most dissolved P and Fe was in colloids >35 nm with apparent molecular weight between 30,000 and 150.000. The bulk of FA occurred in particles <35nm.
3. Calculating the atomic ratios of P, Fe and organic C in the different size classes revealed that Fe-FA chelates are a minor species of the Fe pool. Less than 10% of the fulvic acids occurred as Fe-FA chelates.
4. Based on their apparent size and molecular weight, the Fe-FA chelates are colloidal aggregates, probably with the formula Fe_n-1(FA)_n.
5. About 50% of the dissolved P had the same size as the Fe-FA aggregates. These aggregates were only noticeable in winter when humus-rich polder water was flushed through the lake. During the rest of the year the dissolved Fe and P consisted mainly of acid-labile inorganic colloids which might have been organically coated.     

Proteinase inhibitors in aggregated forms of boar seminal plasma proteins

Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I–V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5–25 kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (M_r>100 kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also β-microseminoprotein (β-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5–25 kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with M_r 8 kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown.     

R-loop hybridization of collagen DNA: Separation in Cs2SO4 gradients

High molecular weight poly(A)⁺ RNA (26–35 S) from chicken embryo trunks which is enriched for collagen mRNA was iodinated and hybridized to DNA under conditions of R-loop formation. The R-loops were separated in Cs₂SO₄ gradients from the bulk of DNA yielding double stranded DNA enriched for collagen genes.     

Immobilization of dissolved organic matter from groundwater discharging through the stream bed

SUMMARY. 1. In laboratory experiments, 9.7–25.7% of dissolved organic carbon (DOC) in groundwater (at concentrations of 18.7–24.8 mg 1-⁻¹) was immobilized after perfusion through 8-cm-deep (22-cm-diameter) cores of stony stream-bed substratum.
2. This represented immobilization rates of 7.1–23.5 mg m⁻² h⁻¹ or, extrapolated across the year, potential immobilization rates within the stream bed of 62.2–205.9g m⁻² yr⁻¹. Actual rates in the entire stream bed were probably higher because perfusion through the experimental cores did not reduce groundwater DOC concentrations to levels measured in the adjacent stream.
3. Natural concentrations of dissolved free amino acids (DFAAs) in groundwater were generally unchanged following perfusion through the cores, suggesting the maintenance of a dynamic equilibrium in their concentrations.
4. Selective enrichments of amino acids in groundwater (up to 20-fold) were entirely immobilized following perfusion, indicating their rapid retention and flux in this environment. Thus, immobilization of the bulk DOC in stream-bed cores probably did not reflect net reductions in dissolved free, low-molecular-weight material, with higher molecular weight, more 'refractory' material being immobilized instead.
5. We conclude that groundwater can contribute substantial amounts of DOC, both high and low molecular weight, to a stream ecosystem. The stream bed is the site at which much of this material could be initially immobilized and made available to the stream trophic structure.     

Inhibitory effect of adrenaline and hydrocortisone on the growth of Allium cepa roots

The roots of Allium cepa were allowed to grow in distilled water containing 10(-4) M adrenaline hydrochloride or 2 X 10(-3) M hydrocortisone sodium succinate. Adrenaline inhibited the growth of roots; they decreased in length, number and total dry weight. The total amount of DNA in the roots was reduced much less than that of extracellular root components after adrenaline. Also hydrocortisone treatment resulted in a considerable decrease of the length and dry weight of Allium cepa roots. Both DNA and extracellular root components were influenced.     

Response of Wolfiporia cocos to iron availability: alterations in growth, expression of cellular proteins, Fe3+-reducing activity and Fe3+-chelators production

Aims:  The main objective of this study was to evaluate the behaviour of the brown-rot fungus Wolfiporia cocos under differential iron availability.
Methods and Results:  W. cocos was grown under three differential iron conditions. Growth, catecholate and hydroxamate production, and mycelial and extracellular Fe³⁺-reducing activities were determined. Iron starvation slowed fungal growth and accelerated pH decline. Some mycelial proteins of low molecular weight were repressed under iron restriction, whereas others of high molecular weight showed positive iron regulation. Mycelial ferrireductase activity decreased as culture aged, while Fe³⁺-reducing activity of low molecular reductants constantly increased. Hydroxamates production suffered only limited iron repression, whereas catecholates production showed to be more iron repressible.
Conclusions:  W. cocos seems to possess more than one type of iron acquisition mechanism; one involving secretion of organic acids and ferrireductases and/or extracellular reductants, and another relying on secretion of catecholates and hydroxamates chelators.
Significance and Impact of the Study:  This paper is the first to report the kinetic study of brown-rot fungus grown under differential iron availability, and the information provided here contributes to address more traditional problems in protecting wood from brown decay, and also makes a contribution in the general area of the physiology of brown-rot fungi.     

Analysis of iron-binding components in the low molecular weight fraction of rat reticulocyte cytosol

Rat reticulocytes were incubated with rat 125I-Tf-59Fe under conditions inhibiting heme synthesis. Cytosol, prepared from the reticulocytes, was separated and analysed by gel filtration and Amicon Ultrafiltration. An iron-containing low molecular weight fraction derived from the cytosol was further analysed by HPLC size-exclusion chromatography and HPLC reversed phase chromatography. Conditions inhibiting heme synthesis and uncoupling the oxidative phosphorylations lead to a large increase in the Fe-containing low molecular weight fraction in the cytosol. The components in the low molecular weight fraction have an apparent molecular weight of 5500 Dalton as determined with HPLC size-exclusion chromatography. The low molecular weight fraction contained several iron chelating components like glycin, 1/2 cystine and citrate, but no specific iron-binding proteins, nucleotides or pyrophosphate.     

Effects of enhanced ultraviolet-B radiation on the production of lipid, polysaccharide and protein in three freshwater algal species

1. The effects of ultraviolet-B (UVB-enhanced) radiation on the production of photosynthates (lipid, protein, polysaccharide and low molecular weight compounds) was examined for three species of algae. Cryptomonas sp., Nitzschia palea and Synechococcus elongatus were selected as representatives of the Cryptophyceae, Bacilliarophyceae and Cyanobacteria, respectively.
2. Laboratory experiments were performed at several UVB_weighted dose rates ranging from 0.018 to 0.391 mW cm^–2. These dose rates span the range of dose rates used in other studies.
3. Effects on the overall photosynthetic rate were observed, even at relatively low UVB_weighted dose rates (0.047 mW cm^–2).
4. The effect of UVB radiation on the fixation of carbon into the main macromolecular pools differed, depending not only on the dosage but on the species examined. However, the observed inhibitory effects were generally non-stochastic. In addition, within each species there were differences in the apparent sensitivity of the various fractions to inhibition by UVB radiation.
5. These results suggest that exposure to UVB radiation has the potential to alter the relative allocation of recently fixed carbon to lipid, protein, polysaccharide and low molecular weight compounds in algae in a species-specific manner.     

The nucleotide sequence for a proline-activating domain of gramicidin S synthetase 2 gene from Bacillus brevis

A fragment encoding proline-activating domain (grs 2-pro) of gramicidin S synthetase 2 (GS 2) was found in an 8.1-kilobase pairs (kb) DNA fragment of Bacillus brevis Nagano, which contained the full length of GS 1 gene (grs 1). The clones designated GS719 and GS708, which expressed gramicidin S synthetase 1, were elucidated to express immunoreactive proteins to GS 2 antibodies with approximate molecular weights of 115,000, 105,000 (GS719), and 110,000 (GS708). The partial purification of the gene products of these clones was carried out using DEAE-Sepharose CL-6B column chromatography. The immunoreactive proteins to GS 2 antibodies were separated from gramicidin S synthetase 1 protein and had specific proline-dependent ATP-32PPi exchange activity. The nucleotide sequence for the proline-activating domain in the 8.1-kb insert was determined. This fragment was 2,879 base pairs long, and encoded 959 amino acids. The calculated molecular weight of 111,671 was consistent with the apparent molecular weight of 115,000 found in SDS-PAGE of the immunoreactive products to GS 2 antibodies. The open reading frame for this protein followed grs 1 gene, though two were separated by a 73-base pair noncoding sequence, and remained open to the end.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation and purification of high molecular weight glycoproteins using agarose gel electrophoresis.

Several components of the extracellular matrix in the molecular weight range of 220 kDa to 150 kDa were purified by preparative electrophoresis on 2.5% Pro-Sieve agarose gels. These high molecular weight glycoproteins, separated under reducing conditions, were recovered in solution by extraction of individual agarose gel slices and analyzed on sodium dodecyl sulfate polyacrylamide gels and Western blots. This simple method permitted the separation and recovery of the laminin B chains (220 kDa and 205 kDa) and entactin (150 kDa) and may prove useful for the purification of other high molecular weight species.     

Colloidal and dissolved organic carbon dynamics in undisturbed boreal forest catchments: a seasonal study of apparent molecular weight spectra

SUMMARY. 1. Colloidal and dissolved organic carbon (CDOC) was monitored in six pristine boreal rivers in Northern Quebec, Canada, using ultrafiltration to obtain apparent molecular weight spectra.
2. The concentration and proportional contribution of small (<1K) molecular weight material (potentially microbially labile) and high (0.7μm to 300 K) molecular weight material was not significantly different between streams of order 2–9.
3. The dominant component of CDOC (0.7μm to 300 K fraction) was probably derived in streams other than first order, from surface or sub-surface run-off, whereas a peak concentration in the <1 K fraction during August was probably due to autochthonous exudates.
4. The two first order streams included in this survey had very low CDOC concentrations. However, further work on twelve other first order streams showed that they had high concentrations and we consider that this is the more typical situation. The finding offered a potential explanation for the otherwise anomalous longitudinal CDOC distribution pattern throughout the study area.