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Biomedical subjects

A Ahmed

Publications and source records attributed to A Ahmed.

At least 19 recordsLinked to original sources

A quantitative secondary structure analysis of the 33 kDa extrinsic polypeptide of photosystem II by FTIR spectroscopy.

In chloroplast photosystem II, the extrinsic polypeptide of 33 kDa is involved in the stabilization the Mn cluster in charge of water splitting and in the fulfilment of the Ca(2+)-cofactor requirement for oxygen evolution. The conformational analysis of the purified 33 kDa extrinsic polypeptide was carried out using FTIR spectroscopy with its self-deconvolution and second derivative resolution enhancement as well as curve-fitting procedures. The FTIR spectroscopic results showed that the isolated polypeptide is characterized by a major proportion beta-sheet conformation (36%) with 27% alpha-helix, 24% turn, and 13% beta-antiparallel structures.

Calcium

Sodium-independent currents of opposite polarity evoked by neutral and cationic amino acids in neutral and basic amino acid transporter cRNA-injected oocytes.

To elucidate the electrical events associated with the movement of amino acids by the neutral and basic amino acid transporter (NBAT)-encoded protein (Yan, N., Mosckovitz, R., Gerber, L.D., Mathew, S., Murty, V.V. V.S., Tate, S.S., and Udenfriend, S. (1994) Proc. Natl. Acad. Sci. USA 91, 7548-7552), we have investigated the membrane potential and current changes associated with the increased transport of amino acids across the cell membrane of NBAT cRNA-injected Xenopus laevis oocytes. Superfusion of 0.05 mM L-phenylalanine, in current-clamped NBAT-injected oocytes, caused a hyperpolarization (8.5 +/- 0.9 mV), but superfusion of L-arginine caused a depolarization (18.3 +/- 1.3 mV). In voltage-clamped (-60 mV) oocytes, superfusion of L-phenylalanine evoked a sodium- and chloride-independent, saturable (Km = 0.34 +/- 0.02 mM, Imax = 31.3 +/- 0.5 nA), outward current. This outward current was reduced in the presence of high external [K] and was barium-sensitive. Outward currents were also evoked by L-leucine, L-glutamine, L-alanine, D-phenylalanine, and L-beta-phenylalanine. Superfusion of L-arginine evoked a saturable (Km = 0.09 +/- 0.02 mM, Imax = -29.2 +/- 1.3 nA) inward current; L-lysine and D-arginine also evoked inward currents. L-Glutamate and beta-alanine failed to evoke any currents. Effluxes of L-[3H]phenylalanine and L-[3H]arginine were trans-stimulated in the presence of either amino acid. Flux-current comparisons indicated amino acid:charge movement stoichiometry of 1:1 for both neutral and cationic amino acids. These findings indicate that the amino acid transport activity(ies) expressed in NBAT cRNA-injected oocytes is electrogenic by a mechanism including the outward movement of a net positive charge (potassium ion or cationic amino acid) in exchange for uptake of a neutral amino acid.

Amino Acid Transport Systems, Basic

Tyrosinase-type prophenoloxidase distribution in the alimentary canal of strains of Anopheles gambiae refractory and susceptible to Plasmodium infection.

Tyrosinase-type prophenoloxidase distribution was investigated in the posterior midgut and salivary glands of strains of noninfected adult Anopheles gambiae refractory (Blue strain) and susceptible (G3 strain) to infection with malaria parasites. Immunocytochemical localization showed that in the posterior midgut epithelium tyrosinase-type prophenoloxidase was almost entirely restricted to apical granules. These are known to be a mixture of secretory granules and lysosomes; it was not possible to distinguish between the two in this study. The secretory product in the lumen of the distal median lobe of the salivary gland was heavily labeled; the secretory product in the lumen of the distal lateral lobes was also labeled but to a lesser extent. Subjectively, no differences in the degree or pattern of labeling of either midgut or salivary glands between the strains of mosquito were apparent.

Animals

A study of recurrent stone formers with special reference to renal tubular acidosis.

Forty-five patients with recurrent renal stone were examined for distal renal tubular acidosis (dRTA) defects by acid challenge test (150 mg ammonium chloride/kg body weight). Their 24-h urine samples were analysed for creatinine, calcium, oxalic acid, inorganic phosphorus, uric acid, magnesium and citric acid. One-hour urine samples before acid load and hourly samples for the 7 h following acid challenge test were collected and analysed for creatinine, calcium, citric acid, inorganic phosphorus, titratable acidity, and ammonium. The incidence of distal RTA defect was 22.2% in the patients examined. The major biochemical characteristics in RTA patients compared with patients without RTA were: (a) significantly higher urinary pH, (b) significantly lower excretion of citric acid, (c) no significant difference in calcium excretion and (d) a tendency toward lower titratable acidity and ammonium excretion.

Acidosis, Renal Tubular

Influence of age and gender on the presence of coronary calcium detected by ultrafast computed tomography.

OBJECTIVES: This study sought to determine the relation between coronary calcification detected with ultrafast computed tomography and lumen narrowing defined with angiography and evaluated whether this relation is influenced by age and gender. BACKGROUND: Ultrafast computed tomography has been shown to be a sensitive method for detection of coronary calcification associated with atherosclerotic disease, but the relation between the extent of coronary calcification and degree of lumen narrowing and the possible influence of gender or age, or both, on this relation have not been clarified. METHODS: Seventy men and 70 women were studied with ultrafast computed tomography for analysis of coronary calcification and coronary angiography. Coronary atherosclerosis was considered present if any lumen irregularity was noted on angiography, and obstructive coronary artery disease was defined as a lumen diameter narrowing > or = 70%. RESULTS: Coronary calcification had a sensitivity of 88% for identification of patients with atherosclerotic disease and 97% for those with obstructive disease, with corresponding specificities of 55% and 41%, respectively. The sensitivity of coronary calcium for detection of atherosclerotic disease in women < 60 years old was 50%, significantly less than the 97% sensitivity in women > 60 years old and the 87% sensitivity in men < 60 years old (p < 0.05 for each comparison). Logistic regression analysis revealed a 1.81-fold increase in the likelihood of detecting coronary calcification in the atherosclerotic lesions of men compared with those in women (95% confidence interval 1.12 to 2.93, p = 0.016) when controlled for age and severity of coronary disease by angiography. CONCLUSIONS: Atherosclerotic lesions in women are less likely to have coronary calcium than lesions with a similar degree of lumen narrowing in men. Differences in the pattern of coronary calcification between men and women may provide insight into the gender differences observed in the clinical development of symptomatic coronary artery disease.

Aged

Renin-producing leiomyosarcoma originating in the uterus.

We present a case of uterine leiomyosarcoma in a 60-year-old woman, who had severe hypertension with hypokalaemia and metabolic alkalosis. Investigation revealed an elevated serum renin level; the tumour stained for renin on immunocytochemistry. The serum renin level fell on successful treatment of the primary. We suggest that this is the first reported case of uterine leiomyosarcoma shown to be producing renin.

Female

Establishment and evaluation of a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex.

OBJECTIVE: To establish a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex and to evaluate the test in the diagnosis of tuberculosis. DESIGN: Three sets of primers were used to amplify 383 bp, 240 bp and 131 bp DNA fragments from the genes encoding the 65 kDa, MPB64 and the 19 kDa proteins of M. tuberculosis in a single reaction tube. Reaction conditions were optimized with respect to the requirement of DMSO, concentration of MgCl2, annealing and denaturation temperatures and number of amplification cycles. Inhibitory activity in clinical samples was identified by amplifying a 500 bp DNA fragment of the phage lambda along with the mycobacterial targets within the same reaction tube. The multiplex PCR was evaluated in differentiating M. tuberculosis complex from other mycobacteria and in the diagnosis of tuberculosis by testing clinical specimens. RESULTS: Amplification of the 383 bp DNA fragments was specific to the genus Mycobacterium. The 240 bp DNA fragment was amplified from M. tuberculosis complex and M. fortuitum and the 131 bp DNA fragment was amplified from the mycobacteria of M. tuberculosis complex and M. scrofulaceum. All the three bands were amplified only from M. tuberculosis complex. Applicability of the multiplex PCR is demonstrated in differentiating M. tuberculosis complex from other mycobacteria by using standard strains and clinical isolates. The multiplex PCR was also useful in the detection of inhibitory activity and in the identification of M. tuberculosis complex directly in clinical samples. CONCLUSION: The multiplex PCR established in this study could differentiate M. tuberculosis complex from other mycobacteria. This test may also be helpful in the early and specific diagnosis of tuberculosis.

Bacterial Typing Techniques

Kinetics of monocyte 1 alpha-hydroxylase in renal failure.

In chronic uremia, the requirement of supraphysiological doses of serum 25-hydroxyvitamin D3 [25(OH)D3] for the normalization of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels has been attributed to impaired substrate availability to renal 1 alpha-hydroxylase. Because serum 1,25(OH)2D3 can also be corrected by 25(OH)D3 supplementation in bilaterally nephrectomized patients, we examined the role of substrate availability on 1,25(OH)2D3 production by peripheral blood monocytes (PBM). In hemodialysis patients (HP), 25(OH)D3 uptake was 50% lower than normal, and the maximal velocity (Vmax) and apparent Michaelis constant (Km) for 25(OH)D3 of 1 alpha-hydroxylase were 2.7- and 4-fold above normal, respectively. When serum 1,25(OH)2D3 of HP was corrected by intravenous 1,25(OH)2D3, 25(OH)D3 uptake, Km, and Vmax returned to normal values. The effect of 25(OH)D3 supplementation was also examined. In normal adults, 25(OH)D3 administration had no effect on serum 1,25(OH)2D3 levels nor on the Km or the Vmax of PBM 1 alpha-hydroxylase but caused a 11-fold increase in serum 24R,25-dihydroxyvitamin D3[24R, 25(OH)2D3]. In HP, 25(OH)D3 therapy raised serum 1,25(OH)2D3 and reduced the Km and Vmax of PBM 1 alpha-hydroxylase, which correlated negatively with serum 1,25(OH)2D3. However, serum 24R,25(OH)2D3 only increased slightly above basal. These results demonstrate that, in HP, 1) impaired uptake of 25(OH)D3 and low affinity for substrate determine the need for high 25(OH)D3 levels to normalize serum 1,25(OH)2D3, despite higher enzymatic activity; 2) 1,25(OH)2D3 deficiency plays a role in enhanced 1,25(OH)2D3 synthesis and impaired access of 25(OH)D3 to PBM 1 alpha hydroxylase; and 3) abnormal 25(OH)D3 delivery also affects 24-hydroxylation.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase

Localization of the angiotensin II and its receptor subtype expression in human endometrium and identification of a novel high-affinity angiotensin II binding site.

Angiotensin (ANG) II is not only a potent vasoconstrictor but may also be involved in the regeneration of new blood vessels. In proliferative endometrium, ANG II-like immunoreactivity was detected in glandular epithelium and stroma with negligible staining around the vascular endothelium. In contrast, in secretory endometrium intense immunostaining was seen in the perivascular stromal cells around the endometrial spiral arterioles with negligible staining of the other cell types. Quantitative receptor autoradiography using the nonselective radioligand [125I]-ANG II and subtype selective competing compounds showed that endometrium contained predominantly AT2 receptors, with relatively low expression of AT1 receptors and a novel non-AT1/non-AT2 angiotensin II recognition site that was insensitive to AT1 or AT2 selective ligands. Levels of specific [125I]-ANG II receptor binding displayed cyclic changes during the menstrual cycle, reaching a maximum in early secretory endometrium and then decreasing in mid to late secretory endometrium to levels seen in early to mid proliferative endometrium. In situ hybridization showed AT1 receptor mRNA expression in the glands and in the endometrial blood vessels. The cyclic changes in ANG II-like immunoreactivity together with expression of both the known and the novel AT receptor subtypes imply that this octopeptide may play a dual role both in the control of the uterine vascular bed and also in the regeneration of the endometrium after endometrial shedding, acting as an angiogenic and mitogenic mediator.

Adult

Development of a lambda-based complementation assay for the preliminary localization of lacI mutants from the Big Blue mouse: implications for a DNA-sequencing strategy.

The Big Blue transgenic mouse carrying the E. coli lacI gene as a mutational target in a lambda-based shuttle vector has been receiving increasing attention in genotoxicity testing because it offers the potential of studying mutation in a mammalian system in vivo. The system not only provides information on mutant frequency, but it also offers the potential of providing information about mutational specificity. Such data is not only important for studies of mutational mechanisms; it offers a critical advantage for determining the mutational response at levels where significant increases in mutant frequency have not been discerned. The repeated sequencing of the entire 1080-bp lacI target, however, remains a formidable task. Here we report on the adaptation of the "negative complementation" assay for the lacI-d phenotype to accommodate the lambda lacI recovered from the Big Blue transgenic animal. This assay permits the localization of mutations to an approximately 330-bp region to facilitate the production of mutational specificity data. The assay is based upon lysogenization of the lambda containing the lacI mutation into a lacI+ host. Of 107 sequenced lacI mutants recovered from Big Blue mice, 74 were identified as NC+ (lacI-d) using this assay. Of these 74, 49 occurred in the region 32-208 bp, which has traditionally been viewed as the NC+ domain. 33 of these mutations were previously identified as producing the NC+ phenotype while another 7 occurred at sites where NC+ mutants have been recovered, but involved a new base substitution. 9 mutants involved new sites. An additional 25 mutants located downstream of the presumed NC+ region were also found to be NC+ as determined by their blue colour on X-gal plates. Of these, 18 occurred in the 209-360-bp region. In parallel, 54 lacI mutants carrying unknown mutations were examined. 37 of these produced blue colonies in this assay. The sequencing of these mutants revealed that 20 (54%) of the 37 mutants were located in the 32-208-bp region. This complementation assay can potentially reduce the amount of DNA sequencing necessary to produce a mutational spectrum by optimising the choice of sequencing primers, and thus provide a significant saving of the material and time required. Furthermore, evidence indicates that the restriction of the mutational target to the NC+ region extends these savings without reducing the usefulness of the mutational specificity data.

Amino Acid Sequence

Vascular endothelial growth factor receptor localization and activation in human trophoblast and choriocarcinoma cells.

Vascular endothelial growth factor (VEGF; also known as vascular permeability factor) is a secreted angiogenic growth factor. It is highly specific for endothelial cells, and its receptor, the fms-like tyrosine kinase (flt), has been localized only to endothelial cells in vivo. Here we describe the expression of mRNA encoding flt in human trophoblast as revealed by in situ hybridization. This mRNA is highly expressed in the cytotrophoblast shell and columns and also highly expressed by the extravillous trophoblast (EVT) in the maternal decidua both in the first trimester and at term. The trophoblast-like choriocarcinoma cell line BeWo also expresses this receptor and the related receptor, kinase domain-containing receptor (KDR), which is also a receptor for VEGF. Treatment of the cell line BeWo with VEGF165 stimulated 3H-thymidine incorporation and tyrosine phosphorylation of MAP (mitogen-activated protein) kinase in a time- and dose-dependent fashion. This study is the first demonstration of the presence of flt on non-endothelial cells in vivo and suggests a role for VEGF in the growth and differentiation of cytotrophoblast at implantation.

Base Sequence

Suicidal inhalation of vehicular exhaust in the Lothian and Borders region of Scotland.

1. A 6-year retrospective study of the features of 79 consecutive completed suicides from exhaust fume inhalation (SEFI) in the Lothian and Borders region of Scotland was conducted. Full forensic autopsies with histological and toxicological studies were carried out. 2. The mean incidence is 2.0/100,000 population (M = 3.7; F = 0.4; P <> 0.001); increasing since 1990. The age peak is in the 35-44 years age group (especially among males) and a significant (P < 0.001) proportion reside in rural areas. 3. SEFI is significantly (P < 0.05) higher during spring and summer; outdoor locations are prevalent during summer. 4. Most (75.6%) of the victims were in current employment. Psychiatric illness (30.7%), problems in marriage or relationships (28.2%) and financial difficulties (16.7%) were the major associated socio-medical factors. 5. The mean carboxyhaemoglobin (COHb) saturation was 74 +/- 11.2%; fatal COHb saturation levels were still detectable in severely decomposed bodies. The blood alcohol concentration (BAC) in 37 of the victims ranged from 14-397 mg dl-1. No significant correlation exists between COHb saturation and the victim's age or BAC. 6. Attempts to reduce the incidence of SEFI must be directed principally to dealing with associated sociomedical problems. The reduction of the potential of vehicles to produce carbon monoxide and of directing exhaust fumes into the interior of the vehicle are important secondary preventative measures.

Adolescent

Colocalization of acidic and basic fibroblast growth factor (FGF) in human placenta and the cellular effects of bFGF in trophoblast cell line JEG-3.

The placenta undergoes extensive angiogenesis and cellular proliferation to establish adequate blood supply to the fetus. The aim of this study was to compare and contrast the immunolocalization of acidic and basic fibroblast growth factor (FGF) in both first trimester and term placenta and gestational decidua. Human choriocarcinoma cell line JEG-3 were employed as a model of cytotrophoblast and the effect of basic FGF on cell proliferation and phospholipase C and D activation investigated. Basic FGF-immunoreactivity (IR) was detected in or around cytotrophoblast cells and in extravillous trophoblast in first trimester placenta by immunohistochemistry using primary polyclonal rabbit antibodies. Identical staining patterns were produced by acidic FGF antibodies indicating colocalization of acidic FGF and basic FGF. At term, weaker and more diffuse staining was seen in the syncytiotrophoblast surrounding the placenta villi and strong staining was present in the smooth muscle cells of mid and large size placental vessels and in some endothelial cells. Endothelial cells and extravillous trophoblast stained strongly within the decidua at first trimester, whereas the glandular epithelium was weakly stained. Basic FGF induced [3H]thymidine incorporation in JEG-3 cells in a dose dependent manner and caused an increase in inosital phosphate accumulation in cells pre-labelled with myo-[3H]inosital at similar concentrations, suggesting a role of phospholipase C in JEG-3 cell proliferation. However, basic FGF failed to stimulate phospholipase D activity in cells pre-labelled with [3H]myristic acid. The detection of acid FGF and basic FGF on both maternal and fetal side of the placenta during early pregnancy suggests a role for FGF in angiogenesis, whereas localisation of the growth factor at term, when extensive angiogenesis has diminished, would indicate that FGF may be associated with more differentiated functions of the trophoblast. The nuclear localization of basic FGF in dividing but not non-dividing placental cells together with the effect of basic FGF on JEF-3 cells, strongly supports a role for basic FGF in cytotrophoblast proliferation in vivo.

Cell Division

Immunolocalisation of vascular endothelial growth factor in human endometrium.

Angiogenesis is an essential component of endometrial regeneration after menses in preparation for implantation. Vascular endothelial growth factor (VEGF) is a secreted angiogenic peptide with mitogenic activity specific for endothelial and trophoblast cells. VEGF-immunoreactivity was detected in glandular epithelium throughout the menstrual cycle by immunohistochemistry, but, showed cyclic variation in the stroma and the blood vessels. During the early proliferative phase, strong staining was seen in the glandular epithelial cells while staining in the stroma was confined to a subpopulation of stromal cells and endometrial blood vessels appeared negative. In contrast, very intense staining of the endometrial stromal cells was seen in the mid proliferative endometrium possibly due to increased synthesis of VEGF by oestrogen. In the late proliferative endometrium, staining was seen in the endothelial cells and the perivascular stromal cells around the endometrial blood vessels. The greatest degree of immunostaining of stromal cells was observed in the mid to late proliferative endometrium. Throughout the secretory phase no staining was seen around the endometrial blood vessels and staining of endometrial stromal cells was confined to early secretory endometrium. In the late secretory endometrium only the glands were positive to VEGF antibody. The observed increase in the immunostaining of stroma suggests increased production of VEGF from early to mid and late proliferative endometrium which parallels the increase in the oestradiol levels in the proliferative phase of the menstrual cycle. It is proposed that VEGF may serve as a paracrine mediator of the effects of ovarian steroids on endometrial vascular development.

Adult

Cell-shape-associated transcriptional activation of the p52(PAI-1) gene in rat kidney cells.

The microfilament-disrupting agent cytochalasin D (CD) increased (by 10-22-fold) the synthesis de novo and extracellular matrix deposition of plasminogen-activator inhibitor type-1 [p52(PAI-1)] in normal rat kidney (NRK) cells. Transition from a flat to a round phenotype occurred concomitantly with, and may actually precede, p52(PAI-1) induction; both the morphological and p52(PAI-1) responses were dose-dependent. Augmented synthesis became evident between 4 and 5 h of treatment of NRK cells with 100 microM-CD, correlating with a transition from 25 to more than 60% rounded cells. CD-associated increases in p52(PAI-1) mRNA abundance and protein biosynthesis were maximal between 6 and 8 h of continuous CD exposure, declined by 50% thereafter, but remained elevated (by at least 6-21-fold respectively over control values) for 24 h. Changes in p52(PAI-1) mRNA abundance at this 24 h point reflected an approx. 5-fold increase in p52(PAI-1)-gene transcription. These data confirm previous suggestions, based on actinomycin D-sensitivity of the inductive response [Higgins & Ryan (1992) Biochem. J. 284, 433-439], that CD-mediated increases in p52(PAI-1) expression are at least partly due to transcription-level events. Since CD also augments specific cellular responses to growth factors or cytokines, the potential effectiveness of this inducer was evaluated both in the presence and absence of serum growth factors using quiescent NRK cells [a growth state in which p52(PAI-1) is not expressed] as a model system. Induction of p52(PAI-1) synthesis and matrix deposition in CD-stimulated quiescent NRK cells was as efficient under growth-factor-deficient conditions as when CD was added simultaneously with serum. CD alone is thus a complete inducer of p52(PAI-1) expression in NRK cells, an observation that supports the contention that cell shape is an important regulatory element in p52(PAI-1)-gene control.

Animals

Phosphorylation of the Rex protein of human T-cell leukemia virus type I.

Rex protein, the posttranscriptional regulator of human T-cell leukemia virus type I (HTLV-I), is required for the control of viral structural protein expression and virus replication. Rex is a phosphoprotein found predominantly in the cell nucleolus, whose function is thought to be regulated by its nucleolar localization and phosphorylation. Therefore, we investigated the in vivo phosphorylation of Rex protein in more detail. Phosphorylation of Rex occurred in all HTLV-I-infected cell lines examined in vivo, primarily at serine residues and to a very small extent at threonine residues. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) led to significant but transient enhancement of the incorporation of [32P]orthophosphate into Rex protein. N-terminal truncation of Rex protein abolished TPA-dependent phosphorylation. Chymotryptic digestion of phosphorylated Rex yielded two phosphopeptides. In vivo phosphorylation sites were identified as serine residues 70 and 177 and threonine residue 174. Serine 70 was a TPA-dependent phosphorylation site within a regulatory domain. We have already shown that the protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) specifically blocked accumulation of viral unspliced gag-pol mRNA. Therefore, the phosphorylation at serine 70 may be involved in the regulation of Rex function in response to extracellular stimuli.

Amino Acid Sequence

Biochemical and immunocytochemical localization of the 'GLUT5 glucose transporter' in human skeletal muscle.

Using biochemical and immunocytochemical techniques, we have assessed both the protein expression and the cellular localization of the GLUT5 transporter in human skeletal muscle. Human muscle membranes, prepared by subcellular fractionation, were subjected to SDS/PAGE and Western-blot analyses using antiserum raised against a specific C-terminal amino acid sequence of the human GLUT5 transporter. GLUT5 was detected as a discrete 49 kDa protein band in a plasma-membrane-enriched fraction prepared from either soleus or gracilis muscle. In contrast, GLUT5 protein was not detectable to any significant extent in fractions which were devoid of muscle plasma membranes (mean GLUT5 abundance in intracellular fractions from three muscle preparations amounted to approximately 10% of that in the plasma-membrane-enriched fraction). Immunofluorescence studies using cryostat sections of human triceps muscle supported the biochemical observations and revealed that GLUT5 antibody selectivity labelled the plasma membrane of muscle cells. This immuno-labelling was significantly suppressed after tissue incubation with antiserum in the presence of a 14-amino-acid synthetic peptide corresponding to a specific C-terminus sequence of human GLUT5. These results indicate that human skeletal muscle expresses the GLUT5 transporter and that it is specifically localized to the plasma membrane, where it may participate in regulating hexose transfer across the sarcolemma.

Blotting, Western