scholarly journals First Record of Pea enation mosaic virus Naturally Infecting Chickpea and Grasspea Crops in Syria

Plant Disease ◽  
2001 ◽  
Vol 85 (9) ◽  
pp. 1032-1032 ◽  
Author(s):  
K. M. Makkouk ◽  
S. G. Kumari ◽  
D.-E. Lesemann
Keyword(s):  
Mosaic Virus ◽  
Acyrthosiphon Pisum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
First Record ◽  
Transmission Efficiency ◽  
Western Blots ◽  
First Report ◽  
Plant Pathol ◽  
Pea Enation Mosaic Virus

Virus-like symptoms not commonly encountered on most chickpea (Cicer arietinum L.) and grasspea (Lathyrus sativus L.) genotypes were noticed at the ICARDA farm near Aleppo, Syria, during April and May 2001. Primary symptoms included stunting, accompanied by leaf mottling and yellowing. The causal agent was transmitted by the pea aphid (Acyrthosiphon pisum Harris) in a persistent manner. Efficiency of transmission was 100% when aphids acquired the virus from grasspea and then inoculated lentil, whereas transmission efficiency was 21% when aphids acquired the virus from chickpea and then inoculated lentil. Samples of symptomatic chickpea and grasspea reacted strongly with the antiserum prepared against a Dutch isolate (E154) of Pea enation mosaic virus (PEMV), provided by L. Bos (Wageningen, the Netherlands) (1), using tissue blot immunoassay (2). Negatively stained preparations from chickpea and grasspea revealed typical PEMV-like isometric particles ≍30 nm in diameter. With immunoelectron microscopy, these particles were effectively trapped and strongly decorated with PEMV antibodies (immunoglobulin G diluted 1:10) provided by M. Musil (Bratislava, formerly Czechoslovakia) (4). The virus capsid protein was 22 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, typical of the PEMV coat protein, and reacted strongly with PEMV antiserum (E154) in western blots. This is the first report of PEMV naturally infecting chickpea and grasspea in Syria and, to our knowledge, the first report in West Asia. PEMV reached epidemic levels on lentil in Syria for the first time in 1994 (3). Field symptoms observed in May 2001 suggest that PEMV may also seriously affect lentil, chickpea, and grasspea crops in Syria. References: (1) K. Mahmood and D. Peters. Neth. J. Plant Pathol. 79:138, 1973. (2) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994. (3) K. M. Makkouk et al. Plant Dis. 83:303, 1999. (4) M. Musil et al. Acta Virol. 14:285, 1970.

scholarly journals First Record of Barley yellow striate mosaic virus Affecting Wheat Summer-Nurseries in Syria

Plant Disease ◽  
2004 ◽  
Vol 88 (1) ◽  
pp. 83-83 ◽  
Author(s):  
Khaled M. Makkouk ◽  
Safaa G. Kumari ◽  
Widad Ghulam ◽  
Nouran Attar
Keyword(s):  
Mosaic Virus ◽  
Barley Yellow Dwarf Virus ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
First Record ◽  
Structural Protein ◽  
Western Blots ◽  
N Protein ◽  
Yellow Dwarf Virus

A limited survey to identify virus diseases affecting wheat in summer nurseries in agricultural stations in southern Syria was conducted during October 2002. A total of 94 bread and durum wheat samples with symptoms suggestive of virus infection (stripping, stunting, and yellowing) were collected. All samples were tested for the presence of four viruses by tissue-blot immunoassay (2) at the Virology Laboratory of ICARDA, Aleppo, Syria using the following polyclonal antibodies: Barley stripe mosaic virus (BSMV); Barley yellow dwarf virus-PAV (BYDV-PAV) and Wheat streak mosaic virus (WSMV) from the Virology Laboratory at ICARDA; and Barley yellow striate mosaic virus (BYSMV) isolated from Italy (BYSMV-Italy) and provided by M. Conti, Instituto di Fitovirologia applicata, Turino, Italy. Serological results obtained indicated that BYSMV was the most commonly encountered virus (78.7%) followed by BYDV-PAV (22.3%), whereas, BSMV and WSMV were not detected in any of the samples tested. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by western blots, purified BYSMV preparations were observed to contain a 47-kDa structural protein typical of the N protein of Rhabdoviruses that reacted strongly with three BYSMV antisera (BYSMV-Italy, BYSMV-Lebanon [4], and BYSMV-Morocco [1]). Samples that reacted with BYSMV antisera were transmitted from wheat to wheat, barley, and oat plants by the planthopper Laodelphax striatella (Fallen) (Hemiptera: family Delphacidae) in a persistent manner, and the major symptoms of BYSMV on cereal crops were stripping and stunting. BYDV-PAV has been reported from Syria earlier (3) but to our knowledge, this is the first report of BYSMV affecting wheat in Syria. References: (1) B. E. Lockhart et al. Plant Dis. 70:1113, 1986. (2) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994. (3) K. M. Makkouk et al. Phytopathol. Mediterr. 28:164, 1989. (4) K. M. Makkouk et al. Plant Dis. 85:446, 2001.

scholarly journals First Report of Pea Enation Mosaic Virus Affecting Lentil (Lens culinaris) in Syria

Plant Disease ◽  
1999 ◽  
Vol 83 (3) ◽  
pp. 303-303 ◽  
Author(s):  
Khaled M. Makkouk ◽  
Safaa G. Kumari ◽  
Bassam Bayaa
Keyword(s):  
Mosaic Virus ◽  
Lens Culinaris ◽  
Acyrthosiphon Pisum ◽  
First Record ◽  
Growth Reduction ◽  
Virus Incidence ◽  
Tissue Blot Immunoassay ◽  
Disease Agent ◽  
Plant Pathol ◽  
Pea Enation Mosaic Virus

Symptoms suggestive of virus infection in lentil (Lens culinaris Medik.) fields in Dara'a in southern Syria have been observed, in epidemic proportions, almost annually since 1994. A similar epidemic was observed on many lentil genotypes at the ICARDA farm, near Aleppo, as well as in other locations in northern Syria during 1998. Symptoms included growth reduction and rolling of leaves, accompanied by mottling with tip wilting or necrosis. Field symptoms were reproduced on lentil cv. Syrian Local upon mechanical inoculation of plants with inoculum from symptomatic field plants. Transmission tests showed that the disease agent can be transmitted from lentil to lentil, pea (Pisum sativum L.), and faba bean (Vicia faba L.) plants by the pea aphid (Acyrthosiphon pisum Harris) in a persistent manner. More than 500 symptomatic lentil plants were collected and tested for the presence of 14 different viruses by the tissue-blot immunoassay (TBIA) (2). Around 80% of the samples reacted only with antiserum to pea enation mosaic virus (PEMV), a Dutch isolate (E1540) provided by L. Bos, Wageningen, The Netherlands (1). Surveys conducted during the 1997/1998 growing season showed that PEMV was widely distributed in the major lentil-growing areas of Syria: some lentil fields had more than 50% virus incidence. This is the first record of PEMV naturally infecting lentil in Syria. References: (1) K. Mahmood and D. Peters. Neth. J. Plant Pathol. 79:138, 1973. (2) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994.

scholarly journals First Record of Barley yellow striate mosaic virus and Cereal yellow dwarf virus-RPV Infecting Wheat in Uzbekistan

Plant Disease ◽  
2001 ◽  
Vol 85 (10) ◽  
pp. 1122-1122 ◽  
Author(s):  
K. M. Makkouk ◽  
S. G. Kumari ◽  
Z. Kadirova ◽  
A. Zueva
Keyword(s):  
Mosaic Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Yellow Dwarf ◽  
Dwarf Virus ◽  
Western Blots ◽  
Cereal Yellow Dwarf Virus ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
Yellow Dwarf Virus

A preliminary survey to identify virus diseases affecting wheat in Uzbekistan was conducted during May 2001. The survey covered 12 wheat fields from 2 cereal-growing regions (Tashkent-Angren and Tashkent-Samarkand). A total of 250 wheat samples with symptoms suggestive of virus infection were collected and tested for the presence of nine viruses by tissue-blot immunoassay (TBIA) (1) at the Virology Laboratory of ICARDA, Aleppo, Syria, using the following antisera: monoclonal antibodies for Cereal yellow dwarf virus-RPV (CYDV-RPV) (ATCC PVAS-669 [American Type Culture Collection, Manassas, VA]) and Barley yellow dwarf virus-MAV (BYDV-MAV) (ATCC PVAS-673); and polyclonal antibodies for BYDV-SGV and BYDV-RMV (3); BYDV-PAV, Barley stripe mosaic virus, and Wheat streak mosaic virus (from Virology Laboratory, ICARDA); Wheat dwarf virus (provided by J. Vacke, Research Institute of Crop Production, Prague, Czeck Republic); and Barley yellow striate mosaic virus (BYSMV) isolated from Lebanon (2). The most common virus present was BYDV-PAV (detected in 12% of the 250 samples tested), followed by BYDV-SGV (10.8%), BYSMV (5.6%), BYDV-RMV (2.4%), BYDV-MAV (2%), and CYDV-RPV (1.2%). CYDV-RPV was detected in three fields; one field was 50 km southeast of Tashkent, and the other two fields were between Tashkent and Samarkand. The majority of BYSMV-positive samples originated from the same field, ≈40 km northeast of Samarkand. Field symptoms of BYSMV-infected plants included yellow flag leaf and stunting. All samples that produced a positive reaction to BYSMV-Lebanon antiserum were tested against four other rhabdovirus antisera: BYSMV-Italy, BYSMV-Morocco, Cereal chlorotic mottle virus, and American wheat striate mosaic virus. Serological tests showed that 100% of the samples reacted strongly with BYSMV-Italy and BYSMV-Morocco. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by western blots, extracts from BYSMV-infected plants were found to contain 66- and 47-kDa structural proteins, typical of G and N proteins of rhabdoviruses, both of which reacted strongly with BYSMV-Italy antiserum. To our knowledge, this is the first report of BYSMV and CYDV-RPV in Uzbekistan. References: (1) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994. (2) K. M. Makkouk et al. Plant Dis. 85:446, 2001. (3) G. N. Webby and R. M. Lister. Plant Dis. 76:1125, 1992.

scholarly journals First Report of Passiflora virus Y Infecting Macroptilium atropurpureum in Taiwan

Plant Disease ◽  
2012 ◽  
Vol 96 (6) ◽  
pp. 918-918 ◽  
Author(s):  
C.-H. Chiang ◽  
Y.-T. Fan ◽  
T.-A. Yu ◽  
Y.-H. Cheng ◽  
Y.-K. Chen
Keyword(s):  
Mosaic Virus ◽  
Soybean Mosaic Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chenopodium Quinoa ◽  
Viral Rna ◽  
Cdna Fragment ◽  
First Report ◽  
Cp Gene ◽  
Macroptilium Atropurpureum

Macroptilium atropurpureum (siratro plants) is a perennial wild legume plant introduced to Taiwan as a forage crop in 1961 (3) and has become a naturalized weed found all over the island. In 2010, siratro plants with virus-like symptoms of mosaic and leaf deformation were observed on the campus of Da-Yeh University in central Taiwan. Flexuous virus-like particles about 750 × 12 nm were observed in the crude sap extracted from symptomatic leaves with a transmission electron microscope. Crude sap was mechanically inoculated to Chenopodium quinoa and local lesions can be observed on inoculated leaves 4 to 5 days after inoculation. Virus was purified from the leaves of inoculated C. quinoa with modified protocols of Gonsalves and Ishii (2). The virus coat protein (CP) consisted of a single major peptide with relative molecular weight of approximately 33 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral RNA extracted from the purified virus was used as a template and was primed with several primer sets corresponding to potyviruses and carlaviruses in reverse transcription-PCR to amplify possible corresponding cDNA fragments. After several attempts, a cDNA fragment of about 1,300 bp could be amplified with the degenerated primer set of BCMNV-F (5′CCDTGGACDGTWGGVATGAC3′) and BCMNV-R (5′CACCAHACCATRAARCCATTCAT3′), which were designed on the basis of the conserved region of the nuclear inclusion b (NIb) and CP genes of some potyviruses including Bean common mosaic necrosis virus, Soybean mosaic virus, Blackeye cowpea mosaic virus, East Asian passiflora virus, and Passion fruit woodiness virus. BLAST analyses showed the amplicon was highly homologous to that of Passiflora virus Y (PaVY). Together with oligo dT, a specific primer (5′GATGACACTCAAATGGCTG3′) corresponding to PaVY CP was used to amplify the cDNA fragment of the most 3′ region of the viral RNA (about 800 bp). The assembled cDNA fragment of 1,958 bp (Accession No. AB679294) contains a partial NIb gene (877 nt), a complete CP gene (819 nt), and the 3′ noncoding region (262 nt). The CP gene shared sequence identities of 89.4 to 98.9% and 92.7 to 98.9% in nucleotide and amino acid, respectively, to that of documented PaVY isolates. PaVY has also been found to be infecting Vigna trilobata, Rhynchosia minima, Clitoria ternatea, and Passiflora foetida in Australia (1). Here we present the first report to our knowledge of PaVY and its infection of siratro (M. atropurpureum) in Taiwan. Additional work is needed to investigate the spread of PaVY and its interaction with other legume plants in Taiwan. References: (1) B. A. Coutts et al. Arch. Virol. 156:1757, 2011. (2) D. Gonsalves and M. Ishii. Phytopathology 70:1028, 1980. (3) Y. Y. Lai et al. J. Taiwan Livestock Res. 42:19, 2009.

scholarly journals First Report of Chickpea Chlorotic Dwarf Virus Infecting Spring Chickpea in Syria

Plant Disease ◽  
2004 ◽  
Vol 88 (4) ◽  
pp. 424-424 ◽  
Author(s):  
S. G. Kumari ◽  
K. M. Makkouk ◽  
N. Attar ◽  
W. Ghulam ◽  
D.-E. Lesemann
Keyword(s):  
Coat Protein ◽  
Faba Bean ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Leaf Roll ◽  
Sodium Dodecyl ◽  
Dwarf Virus ◽  
Western Blots ◽  
First Report ◽  
Beet Western Yellows Virus

During May 2003, a high incidence of symptoms suggestive of virus infection in spring chickpea were observed in many fields in Al-Ghab Valley, Syria, the ICARDA farm (near Aleppo, Syria), as well as in other locations in northern Syria, including the Idleb governorate. Symptoms observed were yellowing, stunting, and necrosis. A total of 1,345 chickpea samples with these symptoms (331 from Al-Ghab Valley, 269 from the ICARDA farm, and 745 from the Idleb governorate) were collected and tested for the presence of five viruses with tissue-blot immunoassay (TBIA) (4) at the Virology Laboratory of ICARDA, using the following antisera: monoclonal antibodies for Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) (1); Bean leafroll virus (BLRV, family Luteoviridae) (4B10) (3); Beet western yellows virus (BWYV, genus Polerovirus, family Luteoviridae [ATCC PVAS-647, American Type Culture Collection, Manassas, VA]); and Soybean dwarf virus (SbDV, family Luteoviridae, [ATCC PVAS-650]) and polyclonal antibodies for Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus, family Geminiviridae, provided by H. J. Vetten, BBA, Braunschweig, Germany). The most common virus present was BWYV (detected in 54.1% of samples tested), followed by CpCDV (19.2%), BLRV (10.2%), and FBNYV (5.5%). SbDV was not detected in any of the samples tested. Using immunosorbent electron microscopy, infected chickpea samples revealed low numbers of geminivirus-like particles after 15 min of incubation on CpCDV antiserum-coated grids. When CpCDV was purified from infected chickpea plants, the virus coat protein was 32 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis typical of CpCDV coat protein (2) and reacted strongly with CpCDV antiserum in western blots. The CpCDV vector in Syria was found to be Orosius albicinctus Distant, and is thought to be similar to Orosius orientalis (Matsumura), the reported vector of CpCDV (2). FBNYV, BWYV, and BLRV infection of chickpea have been previously reported from Syria, but to our knowledge, this is the first report of CpCDV infecting chickpea in Syria. References: (1) A. Franz et al. Ann. Appl. Biol. 128:255, 1996. (2) N. M. Horn et al. Ann. Appl. Biol. 122:467, 1993. (3) L. Katul. Characterization by serology and molecular biology of bean leaf roll virus and faba bean necrotic yellows virus. Ph.D. thesis. University of Gottingen, Germany, 1992. (4) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994.

scholarly journals First report of pea enation mosaic virus 1 infecting Lathyrus ochrus (L.) DC and L. cicera L. in Greece

2021 ◽  
Author(s):  
Kyriaki Sareli ◽  
Konstantinos Gaitanis ◽  
Ioannis T. Tsialtas ◽  
Stephan Winter ◽  
Elisavet K. Chatzivassiliou
Keyword(s):  
Mosaic Virus ◽  
First Report ◽  
Pea Enation Mosaic Virus

scholarly journals Allamanda Mosaic Caused by Cucumber mosaic virus in Taiwan

Plant Disease ◽  
2005 ◽  
Vol 89 (5) ◽  
pp. 529-529 ◽  
Author(s):  
Y. K. Chen ◽  
C. C. Yang ◽  
H. T. Hsu
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chenopodium Quinoa ◽  
Rabbit Antiserum ◽  
Nucleotide Sequence Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Indicator Plants

Allamanda (Allamanda cathartica L., family Apocynaceae) is native to Brazil and is a popular perennial shrub or vine ornamental in Taiwan. Plants showing severe mosaic, rugosity, and leaf distortion symptoms on leaves are common in commercial nurseries and private gardens. Examination of crude sap prepared from symptomatic leaves using an electron microscope revealed the presence of spherical virus particles with a diameter of approximately 28 nm. The virus was mechanically transmitted to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). The virus caused local lesions on inoculated leaves of Chenopodium quinoa and C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, Nicotiana benthamiana, N. glutinosa, N. rustica, and N. tabacum. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Tests of leaf sap extracted from naturally infected allamanda and inoculated indicator plants using enzyme-linked immunosorbent assay were positive to rabbit antiserum prepared to CMV. Viral coat protein on transblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis reacted with CMV subgroup I specific monoclonal antibodies (2). With primers specific to the 3′-half of RNA 3 (1), amplicons of an expected size (1,115 bp) were obtained in reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from infected allamanda and N. benthamiana. The amplified fragment (EMBL Accession No. AJ871492) was cloned and sequenced. It encompasses the 3′ part of the intergenic region of RNA 3 (158 nt), CP ORF (657 nt), and 3′ NTR (300 nt) showing 91.8–98.9% and 71.4–72.8% identities to those of CMV in subgroups I and II, respectively. Results of MspI-digested restriction fragment length polymorphism patterns of the RT-PCR fragment and the nucleotide sequence analysis indicate that the CMV isolate from allamanda belongs to subgroup IB, which is predominant on the island. To our knowledge, CMV is the only reported virus that infects allamanda and was first detected in Brazil (3), and this is the first report of CMV infection in allamanda plants occurring in Taiwan. References: (1) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (2) H. T. Hsu et al. Phytopathology 90:615, 2000. (3) E. W. Kitajima. Acta. Hortic. 234:451, 1988.

Isolation, Cultivation, and Characterization of Borrelia burgdorferi from Rodents and Ticks in the Charleston Area of South Carolina

2000 ◽  
Vol 38 (1) ◽  
pp. 120-124
Author(s):  
J. H. Oliver ◽  
K. L. Clark ◽  
F. W. Chandler ◽  
L. Tao ◽  
A. M. James ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Borrelia Burgdorferi ◽  
Reference Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Cotton Rat ◽  
Cotton Mouse ◽  
B 31

ABSTRACT Twenty-eight Borrelia burgdorferi isolates from the Charleston, S.C., area are described. This represents the first report and characterization of the Lyme disease spirochete from that state. The isolates were obtained from December 1994 through December 1995 from the tick Ixodes scapularis , collected from vegetation, and from the rodents Peromyscus gossypinus (cotton mouse), Neotoma floridana (eastern wood rat), and Sigmodon hispidus (cotton rat). All isolates were screened immunologically by indirect immunofluorescence with monoclonal antibodies to B. burgdorferi -specific outer surface protein A (OspA) (antibodies H5332 and H3TS) and B. burgdorferi -specific OspB (antibodies H6831 and H614), a Borrelia (genus)-specific antiflagellin antibody (H9724), Borrelia hermsii -specific antibodies (H9826 and H4825), and two polyclonal antibodies (one to Borrelia species and another to B. burgdorferi ). Six of the isolates were analyzed by exposing Western blots to monoclonal antibodies H5332, H3TS, H6831, and H9724. All isolates were also analyzed by PCR with five pairs of primers known to amplify selected DNA target sequences specifically reported to be present in the reference strain, B. burgdorferi B-31. The protein profiles of six of the isolates (two from ticks, one from a cotton mouse, two from wood rats, and one from a cotton rat) also were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the 28 Charleston isolates are B. burgdorferi sensu stricto based on their similarities to the B. burgdorferi B-31 reference strain.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

Degradation artefacts during sample preparation for sodium dodecyl sulphate polyacrylamide gel electrophoresis

1987 ◽  
Vol 7 (3) ◽  
pp. 209-215
Author(s):  
E. Jane Cookson ◽  
Robert J. Beynon
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Glycogen Phosphorylase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Peptide Sequence ◽  
Western Blots ◽  
Breakage Point ◽  
Degradation Intermediates

Preparation of samples for sodium dodecyl sulphate polyacrylamide gel electrophoresis routinely involves heating the protein in solution containing detergent and reducing agent for at least two minutes. Here we show that this treatment causes fragmentation of the protein glycogen phosphorylase, whether purified or as a component of a skeletal muscle preparation. The fragments are detected as minor bands on western blots and represent the products of discrete breakage point in the peptide sequence. Protease inhibitors cannot suppress the fragmentation. Such small amounts of immunoreactive fragments may be incorrectly identified on western blots as contaminants that were originally present in the antigen preparation. They may also be a source of ambiguity in studies that search for degradation intermediates during proteolysis.

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