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At least 955 records · Page 53Linked to original sources
PubMed2002

Interaction of STOP with neuronal tubulin is independent of polyglutamylation.

In eukaryotes, the coordinated progress of the various cellular tasks along with the assembly of adapted cytoskeletal networks requires a tight regulation of the interactions between microtubules and their associated proteins. Polyglutamylation is the major post-translational modification of neuronal tubulin. Due to its oligomeric structure, polyglutamylation can serve as a potentiometer to modulate binding of diverse MAPs. In addition, it can exert a differential mode of regulation towards distinct microtubule protein partners. To find out to what extent polyglutamylation is a general regulator, we have analyzed its ability to affect the binding of STOPs, the major factors that confer cold- and nocodazole-resistance to microtubules. We have shown by blot overlay experiments that binding of STOP does not depend on the length of the polyglutamyl chains carried by tubulins. And contrary to the other microtubule-associated proteins tested so far, STOP can bind quantitatively to any tubulin isoform whatever its degree of polyglutamylation.

Animals↗
PubMed1971

Functional inactivation and appearance of breaks in RNA chains caused by gamma irradiation of Escherichia coli ribosomes.

70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D(37) values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 x 10(5) and 1.4 x 10(5), were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 x 10(5)) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.

Adenine↗
PubMed2006

Temperature-sensitive pluronic/poly(ethylenimine) nanocapsules for thermally triggered disruption of intracellular endosomal compartment.

Pluronic hydrogel nanoparticles cross-linked with poly(ethylenimine) (PEI) were synthesized by a modified emulsification/solvent evaporation method. Pluronic F-127 preactivated at the terminal group with p-nitrophenyl chloroformate was dissolved in dichloromethane, and the organic solution was emulsified in deionized water containing PEI by sonication. Primary amine groups of PEI in the aqueous phase were conjugated and/or cross-linked with activated Pluronic F-127 in the vicinity of the water/dichloromethane interface, resulting in the formation of shell-cross-linked Pluronic/PEI nanocapsules. Pluronic/PEI nanocapsules exhibited a volume transition behavior over a temperature range of 24-33 degrees C. The thermally reversible swelling/deswelling of Pluronic/PEI nanocapsules was caused by temperature-dependent hydrophobic interaction of cross-linked and/or grafted Pluronic polymer chains in the nanocapsules. Pluronic/PEI nanocapsules were utilized to break up intracellular endosomal compartments by swelling-induced destabilization of the endosomal membrane triggered by a cold-shock treatment.

Biocompatible Materials↗
PubMed1991

Purification and characterization of the heat-labile toxin of Bordetella pertussis.

A procedure is described for purification of pertussis heat-labile toxin (PEHLT) from cells of Bordetella pertussis. The purification procedure, performed in the cold and in the presence of protease inhibitors, gives 1,350-fold purification with yields of about 60%. The toxin was shown to be a single-chain polypeptide of 140 kDa, pI 6.02. It was completely inactivated by heating at 56 degrees C for 60 min. Rabbit antiserum prepared against PEHLT neutralized the toxin and gave a single precipitin line on immunodiffusion. In immunodiffusion assays, this anti-PEHLT serum did not react with pertussis toxin, filamentous hemagglutinin, or preparations of pertussis adenylate cyclase. Purified PEHLT elicited dermonecrosis and atrophy of the spleen. PEHLT is extraordinarily active; 0.4 X 10(-12) g caused necrotic lesions in newborn mice, and with 18- to 20-g mice the 50% lethal dose was about 11 X 10(-9) g.

Adenylyl Cyclases↗
PubMed1993

Cervicothoracic sympathectomy for Raynaud's syndrome.

Raynaud's syndrome is a clinical entity characterized by episodic vascular spasm and digital ischemia occurring in response to cold or emotional stimuli. Most patients with Raynaud's syndrome are successfully managed with medical therapy. Surgical sympathectomy is rarely performed and its use remains controversial. Of 3,219 patients seen with the diagnosis of Raynaud's disease, phenomenon or syndrome over the last 10 years at Mayo Clinic, 20 cervical or thoracic sympathectomies were performed in 14 (0.4%) patients. Of all patients who underwent cervico thoracic sympathectomy (68) for various reasons during the same time period, 20.5% (14/68) were performed for Raynaud's syndrome. Surgical sympathectomy was reserved for those rare patients with persistent, severe symptoms (pain, ulcer) despite intensive medical treatment. Surgical therapy was also used for those few patients with occupations involving exposure to cold and disabling, refractory symptoms in hopes of allowing them to return to work. Transaxillary (12 limbs) or supraclavicular (8 limbs) exposure and resection of the sympathetic chain with T1-T4 ganglions was usually performed. Initial resolution or improvement of symptoms was achieved in 19/20 (95%) of limbs. Both patients with digital ulceration healed. Although improved in some, persistent or recurrent symptoms were present in all patients after six months postoperatively. Increased sensitivity of digital vessels to circulating catecholamines, nerve fiber regeneration or incomplete sympathectomy have been postulated to lead to recurrence. Five patients developed Horner's syndrome postoperatively. A portion of the stellate ganglion was intentionally resected in 3 of the 5 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Mapping of four light chain-associated idiotopes of a human monoclonal rheumatoid factor.

In an effort to analyze both IgM rheumatoid factor (RF) repertoire and regulation of RF production in humans, we developed a panel of four mouse monoclonal antibodies (mAb) defining distinct K light chain-associated idiotopes (id) of a human monoclonal IgM RF (Alt). These mAb (A75, AM1, AM2, AM3) had equivalent reactivities with the immunizing RF during classic inhibition of antigen-binding assays. These anti-id reagents were reacting to neither other tested monoclonal IgM RF nor normal polyclonal IgM. It was possible to distinguish the id defined by the mAb from the results of four sets of experiments: dissociation of Alt RF heavy (H) and light (L) chains showed that A75, AM1, and AM2 reacted to id located on the L chain, whereas AM3 defined a conformational RF id; recombination experiments of H and L chains showed that A75 and AM2 reacted well with both homologous (Alt H + Alt L) and heterologous (Alt L + unrelated H) recombinants, whereas AM1 reacted better with the homologous recombinant than with the heterologous one; the relative affinities of the mAb were drawn from their ability to shift already bound labeled Alt RF from solid phase IgG; and radiolabeling of two mAb (A75 and AM3) and experiments of inhibition of id binding with cold anti-id and cold anti-CK showed that A75 recognized a proximal id (close to the K constant region), whereas AM3 defined a more distal id, AM2 and AM1 being located between A75- and AM3-defined sites. This topographic mapping of K light chain-associated id of a human RF with anti-id of known relative affinities could help in studying idiotypic regulation in humans.

Antibodies, Monoclonal↗
PubMed2004

Role of membrane lipid fatty acids in cold adaptation.

Psychrophilic and psychrotolerant bacteria have evolved various strategies to adapt to low temperature. One important strategy, which is crucial to the survival of the cell at low temperature, relates to the ability of the cell to modulate the fluidity of the membrane. Bacteria in general modulate membrane fluidity by altering their fatty acid composition. But, bacteria could also achieve the same by various other strategies such as by altering the lipid head group, the protein content of the membrane, the type of carotenoids synthesized, the fatty acid chain length and the proportion of cis to trans fatty acids. In addition bacteria have a two-component signal transduction pathway consisting of a membrane-bound sensor and a soluble cytoplasmic response regulator involved in the perception and transduction of low temperature signals. This review on cold adaptation highlights the various strategies by which bacteria modulate the fluidity of the membrane and the process by which it senses and transduces the low temperature signal.

Adaptation, Physiological↗
PubMed2006

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking.

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.

DNA, Fungal↗
PubMed2003

Coronavirus 229E-related pneumonia in immunocompromised patients.

Coronaviruses strains 229E and OC43 have been associated with various respiratory illnesses ranging from the self-resolving common cold to severe pneumonia. Although chronic underlying conditions are major determinants of severe respiratory virus infections, few data about coronavirus-related pneumonia in immunocompromised patients are available. Here we report 2 well-documented cases of pneumonia related to coronavirus 229E, each with a different clinical presentation. Diagnosis was made on the basis of viral culture and electron microscopy findings that exhibited typical crown-like particles and through amplification of the viral genome by reverse transcriptase-polymerase chain reaction. On the basis of this report, coronaviruses should be considered as potential causative microorganisms of pneumonia in immunocompromised patients.

Adolescent↗
PubMed1975

Kinectics of beta-galactosidase synthesis in Escherichia coli at 5 C.

The defect in protein synthesis that is observed in Escherichia coli after transfer to low temperature was studied. For the enzyme beta-galactosidase, the elongation reactions of transcription and translation can take place slowly but normally at 5 C. The time necessary to complete the coupled synthesis of the beta-galactosidase messenger ribonucleic acid and polypeptide chain was found to be about 80 min at 5 C. From this result and from the known length of the beta-galactosidase monomer, it is possible to calculate that at 5 C one amino acid is added to the growing polypeptide chain every 4 s. The initiation of transcription of the beta-galactosidase messenger is inhibited after transfer to 5 C. This fact alone, however, cannot account for all of the phenomena observed at 5 C, because a given amount of messenger yields less enzyme at 5 C than it does at 37 C. Furthermore, in cells induced for short periods at 37 C, the capacity to synthesize beta-galactosidase after transfer to 5 C was found to accumulate linearily with the square of the time of induction. Two alternative models could account for these data. If all ribosomes that initiate translation at 37 C yield complete beta-galactosidase polypeptide chains at 5 C, then an inhibition of translation initiation after transfer to 5 C must be invoked to explain the results. If, on the other hand, a substantial portion of the ribosomes that initiate translation at 37 C do not yield complete beta-galactosidase polypeptides at 5 C, then intracistronic polarity could account for the data, and there is no need to invoke an inhibition of translation initiation at 5 C.

Bacterial Proteins↗
PubMed2005

An evaluation of disinfectants for the sanitation of porcine reproductive and respiratory syndrome virus-contaminated transport vehicles at cold temperatures.

The objective of this study was to evaluate the efficacy of commercially available disinfectants to sanitize porcine reproductive and respiratory syndrome virus (PRRSV) contaminated trailer models in cold climates (-20 degrees C and 4 degrees C). Disinfectants evaluated included Synergize, Aseptol 2000, Biophene, Sentramax, Virkon, Tek Trol, and DC&R. All products were applied to trailers via fumigation at 4 degrees C. Following experimental contamination of model trailers with PRRSV MN 30-100 (5 x 10(5) TCID50), models were tested for the presence or absence of PRRSV-RNA by polymerase chain reaction (PCR) on swabs collected 0, 30, and 60 min after treatment. Treatments included washing only, washing plus disinfectant fumigation, washing plus fumigation, and washing plus overnight drying. The PRRSV-RNA detected across trailers ranged from 0/12 replicates in trailers treated with Synergize or allowed to dry for 8 h. These trailers were also negative for the presence of infectious PRRSV, based on the lack of sentinel pig infection (0/4 replicates). In contrast, the detection of PRRSV-positive swabs by PCR ranged from 3/12 (Aseptol) to 10/12 (Biophene). Based on these results, the efficacy of Synergize was evaluated at -20 degrees C. In an attempt to reduce the impact of freezing on disinfectant activity, 30 mL of disinfectant was added to a 3840 mL of a 40% methanol solution, a 10% propylene glycol (PG) solution, or water alone. The PRRSV-contaminated trailers were treated with 1 of 3 disinfectant mixtures via fumigation, stored for 8 h at -20 degrees C, allowed to thaw, and sampled as described. Trailers treated with 40% methanol or 10% PG did not freeze and were negative for PRRSV-RNA and infectious virus following thawing. In contrast, trailers treated with disinfectant and water were frozen within 60 min at -20 degrees C, and decontamination was not successful.

Animals↗
PubMed2004

Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability.

The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5-25 degrees C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t(1/2) value of 16 min for thermal inactivation during incubation at 40 degrees C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly(244) was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly(244)-->Pro substitution into the enzyme. Stability studies showed that the t(1/2) value for thermal inactivation of the mutant during incubation at 40 degrees C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k(cat)/K(m) value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.

Amino Acid Sequence↗
PubMed2003

cor Gene expression in barley mutants affected in chloroplast development and photosynthetic electron transport.

The expression of several barley (Hordeum vulgare) cold-regulated (cor) genes during cold acclimation was blocked in the albino mutant a(n), implying a chloroplast control on mRNAs accumulation. By using albino and xantha mutants ordered according to the step in chloroplast biogenesis affected, we show that the cold-dependent accumulation of cor14b, tmc-ap3, and blt14 mRNAs depends on plastid developmental stage. Plants acquire the ability to fully express cor genes only after the development of primary thylakoid membranes in their chloroplasts. To investigate the chloroplast-dependent mechanism involved in cor gene expression, the activity of a 643-bp cor14b promoter fragment was assayed in wild-type and albino mutant a(n) leaf explants using transient beta-glucuronidase reporter expression assay. Deletion analysis identified a 27-bp region between nucleotides -274 and -247 with respect to the transcription start point, encompassing a boundary of some element that contributes to the cold-induced expression of cor14b. However, cor14b promoter was equally active in green and in albino a(n) leaves, suggesting that chloroplast controls cor14b expression by posttranscriptional mechanisms. Barley mutants lacking either photosystem I or II reaction center complexes were then used to evaluate the effects of redox state of electron transport chain components on COR14b accumulation. In the mutants analyzed, the amount of COR14b protein, but not the steady-state level of the corresponding mRNA, was dependent on the redox state of the electron transport chain. Treatments of the vir-zb63 mutant with electron transport chain inhibitors showed that oxidized plastoquinone promotes COR14b accumulation, thus suggesting a molecular relationship between plastoquinone/plastoquinol pool and COR14b.

Acclimatization↗
PubMed1995

An alpha tubulin mutation suppresses nuclear migration mutations in Aspergillus nidulans.

Microtubules and cytoplasmic dynein, a microtubule-dependent motor, are required for nuclei to move along the hyphae of filamentous fungi. Nuclear migration in Aspergillus nidulans is blocked by heat-sensitive (hs-) mutations in the nudA gene, which encodes dynein heavy chain, and the nudF gene, which encodes a G protein beta-subunit-like protein. Hs- mutations in the nudC and nudG genes also prevent nuclear migration. We have isolated extragenic suppressor mutations that reverse the hs- phenotypes caused by these mutations. Here we show that one nudF suppressor also suppresses hs- mutations in nudA, nudC, and nudG and deletions in nudA and nudF. This suppressor mutation is in the tubA alpha tubulin gene, and its characteristics suggest that it destabilizes microtubules. The mutation alters microtubule staining and confers sensitivity to cold and benomyl, two treatments that destabilize microtubules. Treatment with low concentrations of benomyl also suppresses the hs- nudA, nudC, nudF, and nudG mutations and the nudA and nudF deletions. Suppression of the hs- nudA mutation and the nudA deletion is especially interesting because these strains lack active dynein heavy chain. Together, these results suggest that microtubule destabilization allows nuclei to migrate even in the absence of cytoplasmic dynein motor function.

Aspergillus nidulans↗
PubMedDate not supplied

Assessment of myocardial damage by circulating cardiac myosin light chain I after heart transplantation.

The purpose of this study was to evaluate the cardiac damage by cardiac myosin light chain I after transplantation. This study included 30 patients who underwent cardiac operations and who were divided into three groups. These groups consisted of (1) control group, 15 valvular patients without coronary disease (no electrocardiography changes and creatine kinase MB isoenzyme of 100 micrograms/L or less); (2) infarction group, eight patients (six coronary bypass and two valvular patients with perioperative infarction pattern in the electrocardiography and creatine kinase MB isoenzyme of 100 micrograms/L or more; and (3) transplantation group, seven transplant patients (six heart and one heart-lung). The peak cardiac myosin light chain I value in the transplantation group (32.9 +/- 3.4 micrograms/L) was comparable to the infarction group (27.6 +/- 2.6 micrograms/L), and both of them were significantly higher than the control group (9.2 +/- 0.9 micrograms/L) (p < 0.01). Peak cardiac myosin light chain I levels in the control and transplantation groups correlated with the ischemic time (r = 0.48, p < 0.05 and r = 0.67, p < 0.05, respectively). The total dose of dopamine in the transplantation group correlated with the peak cardiac myosin light chain I (r = 0.67, p < 0.05), and with the cardiac myosin light chain I value on day 7 (r = 0.88, p < 0.01). This study suggests that circulating cardiac myosin light chain I estimations are useful to evaluate myocardial damage after transplantation during postoperative week 1.

Aged↗
PubMed1997

Regulation of the elongation-termination decision at intrinsic terminators by antitermination protein N of phage lambda.

The mechanisms that control N-protein-dependent antitermination in the phage lambda life cycle have counterparts in the regulatory systems of other organisms. Here we examine N-dependent antitermination at the intrinsic tR' terminator of lambda to elucidate the regulatory principles involved. The tR' terminator consists of a sequence of six base-pairs along the template at which the transcription complex is sufficiently destabilized to make RNA release possible. Within this "zone of opportunity" for termination the termination efficiency (TE) at each template position is determined by a kinetic competition between alternative reaction pathways that lead either to elongation or to termination. TE values at each position within tR' have been mapped as a function of NTP concentration, and it is shown that N protein (in the presence of NusA and a nut site; the minimal system for N-dependent antitermination) can offset increases in TE that are induced by limiting the concentrations of each of the next required NTPs. By limiting NTP concentrations or working at low temperature we show that a significant effect of N within the minimal system is to increase the rate of transcript elongation three- to fivefold at most positions along the template. Assuming that a comparable increase in elongation rate applies at template positions within the terminator, we show that an increase of this magnitude is not sufficient to account for the antitermination efficiency observed and that an approximately 100-fold stabilization of the transcription complex at intrinsic termination sites as a consequence of binding the N-containing antitermination sub-assembly must be invoked as well. A general method for partitioning TE effects in antitermination between changes in elongation rate and termination complex stability is demonstrated, based on competing free energy of activation barriers for the elongation and termination reactions. The analysis and utility of such mixed modes of transcriptional regulation are considered in general terms.

Bacterial Proteins↗
PubMed1989

Cryocrystalglobulinemia: pH-dependent precipitation of a monoclonal IgG-kappa-immunoglobulin.

Cryoglobulinemia is seen in a minority of patients with plasma cell dyscrasias and can be of clinical relevance if intravascular gelling or precipitate formation occurs at low temperatures. We observed a patient suffering from IgG-kappa multiple myeloma which was complicated by instability of the immunoglobulin forming crystalline precipitates at low pH and low temperature. Short exposure to extreme cooling initiated an unusual course terminating in disseminated vascular occlusion and fatal outcome which was connected with an adverse effect of blood exchange. Crystal formation was noticed in anticoagulated blood samples even at 37 degrees C. In vitro studies showed a critical pH dependency of solubility of the immunoglobulin close to the physiological pH of the blood. These observations suggest that the fatal outcome was due to a vicious circle of ischemia, metabolic acidosis and intravascular precipitations, initiated not only by low acral temperatures but by cold-induced ischemic tissue acidosis as well. Serum of patients with monoclonal gammopathy and cryoglobulinemia should be tested for pH dependent immunoglobulin insolubility.

Aged↗
PubMed2006

Genetic analysis of polynucleotide phosphorylase structure and functions.

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.

Blotting, Northern↗