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

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.

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

Plant Disease ◽

10.1094/pdis.2004.88.1.83a ◽

2004 ◽

Vol 88 (1) ◽

pp. 83-83 ◽

Cited By ~ 8

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.

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

Plant Disease ◽

10.1094/pdis.2001.85.10.1122b ◽

2001 ◽

Vol 85 (10) ◽

pp. 1122-1122 ◽

Cited By ~ 2

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.

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

Plant Disease ◽

10.1094/pdis.2001.85.9.1032c ◽

2001 ◽

Vol 85 (9) ◽

pp. 1032-1032 ◽

Cited By ~ 3

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.

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Triticum mosaic virus: A New Virus Isolated from Wheat in Kansas

Plant Disease ◽

10.1094/pdis-92-5-0808 ◽

2008 ◽

Vol 92 (5) ◽

pp. 808-817 ◽

Cited By ~ 64

Author(s):

Dallas L. Seifers ◽

T. J. Martin ◽

Tom L. Harvey ◽

John P. Fellers ◽

James P. Stack ◽

...

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Polyclonal Antibodies ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cesium Chloride ◽

High Plains ◽

Flight Mass Spectrometry ◽

Sds Page ◽

Systemic Symptoms

In 2006, a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat plants with mosaic symptoms. The physiochemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sequencing of the nucleotides and amino acids of the coat protein, and immunological reactivity. Purified preparations contained flexuous, rod-shaped particles that resembled potyviruses. The coat protein was estimated from SDS-PAGE to have a mass of approximately 35 kDa. Its amino acid sequence, as deduced from DNA sequencing of cloned, reverse-transcribed viral RNA and separately determined by time-of-flight mass spectrometry, was most closely related (49% similarity) to Sugarcane streak mosaic virus, a member of the Tritimovirus genus of the family Potyviridae. The virus gave strong positive reactions during enzyme-linked immunosorbent assays using polyclonal antibodies raised against purified preparations of the cognate virus but gave consistent negative reactions against antibodies to Wheat streak mosaic virus (WSMV), other wheat potyviruses, and the High Plains virus. When the virus was inoculated on the WSMV-resistant wheat cv. RonL, systemic symptoms appeared and plant growth was diminished significantly in contrast with WSMV-inoculated RonL. Taken together, the data support consideration of this virus as a new potyvirus, and the name Triticum mosaic virus (TriMV) is proposed.

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Protein phosphatase PP2C in the flagellum of Leishmania major: cloning and characterization

Parasitology Open ◽

10.1017/pao.2017.14 ◽

2017 ◽

Vol 3 ◽

Cited By ~ 6

Author(s):

A. R. Escalona-Montaño ◽

R. Pérez-Montfort ◽

N. Cabrera ◽

R. Mondragón-Flores ◽

D. E. Vélez-Ramírez ◽

...

Keyword(s):

Protein Tyrosine Phosphatase ◽

Protein Phosphatase ◽

Protein Concentration ◽

Leishmania Major ◽

Polyclonal Antibodies ◽

Divalent Cations ◽

Tyrosine Phosphatase ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Phosphatase Inhibitors

AbstractThe main goal of this work consisted in cloning, purifying and characterizing a protein phosphatase 2C (PP2C) from promastigotes ofLeishmania major. The gene was cloned and amplified by PCR using specific oligonucleotides and the recombinant protein was purified by affinity chromatography. The peak with maximal protein concentration was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and revealed a protein of 44·9 kDa with PP2C activity. This activity was dependent on divalent cations (Mg+2and Mn+2) and was optimal at pH of 8·5, using phosphothreonine as the substrate. Sanguinarine inhibited the activity of the recombinantLmPP2C, while protein tyrosine phosphatase inhibitors had no effect. The recombinantLmPP2C was used to generate polyclonal antibodies. These antibodies recognized a protein of 44·9 kDa in differentLeishmaniaspecies; theLmPP2C was localized in the flagellar pocket and the flagellum of promastigotes.

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First Report of Raspberry bushy dwarf virus in Rubus multibracteatus from China

Plant Disease ◽

10.1094/pdis.2003.87.5.603a ◽

2003 ◽

Vol 87 (5) ◽

pp. 603-603 ◽

Cited By ~ 13

Author(s):

C. J. Chamberlain ◽

J. Kraus ◽

P. D. Kohnen ◽

C. E. Finn ◽

R. R. Martin

Keyword(s):

Amino Acid ◽

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

Dwarf Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Black Raspberry ◽

First Report ◽

Raspberry Bushy Dwarf Virus ◽

Plant Pathol

Raspberry bushy dwarf virus (RBDV), genus Idaeovirus, has been reported in commercial Rubus spp. from North and South America, Europe, Australia, New Zealand, and South Africa. Infection can cause reduced vigor and drupelet abortion leading to crumbly fruit and reduced yields (3,4). In recent years, Rubus germplasm in the form of seed, was obtained on several collection trips to The People's Republic of China to increase the diversity of Rubus spp. in the USDA-ARS National Clonal Germplasm Repository, (Corvallis, OR). Before planting in the field, seedlings were tested for the presence of RBDV, Tomato ringspot virus, and Tobacco streak virus using triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) (antiserum produced by R. R. Martin). One symptomless plant of R. multibracteatus H. Lev. & Vaniot (PI 618457 in USDA-ARS GRIN database), from Guizhou province in China, tested positive for RBDV (RBDV-China). After mechanical transmission on Chenopodium quinoa Willd., this isolate produced typical symptoms of RBDV (3). To determine if RBDV-China was a contaminant during the handling of the plants, or if the source was a seedborne virus, the coat protein gene was sequenced and compared to published sequences of RBDV. RNA was extracted from leaves of R. multibracteatus and subjected to reverse transcription-polymerase chain reaction (RT-PCR) using primers that flank the coat protein gene. Products from four separate PCR reactions were sequenced directly or were cloned into the plasmid vector pCR 2.1 (Invitrogen, Carlsbad, CA) and then sequenced. The coding sequence of the coat protein gene of RBDV-China was 87.5% (722/825) identical to that isolated from black raspberry (Genbank Accession No. s55890). The predicted amino acid sequences were 91.6% (251/274) identical. Previously, a maximum of five amino acid differences had been observed in the coat proteins of different RBDV strains (1). The 23 differences observed between RBDV-China and the isolate from black raspberry (s55890) confirm that the RBDV in R. multibracteatus is not a greenhouse contaminant but is indeed a unique strain of RBDV. In addition, monoclonal antibodies (MAbs) to RBDV (2) were tested against RBDV-China. In these tests, MAb D1 did not detect RBDV-China, whereas MAb R2 and R5 were able to detect the strain. This is the first strain of RBDV that has been clearly differentiated by MAbs using standard TAS-ELISA tests. Although RBDV is common in commercial Rubus spp. worldwide, to our knowledge, this is the first report of RBDV in R. multibracteatus, and the first report of RBDV from China. The effects of this new strain of RBDV could be more or less severe, or have a different host range than previously studied strains. It is more divergent from the type isolate than any other strain that has been studied to date. Phylogenetic analysis of coat protein genes of RBDV may be useful in understanding the evolution and spread of this virus. References: (1) A. T. Jones et al. Eur. J. Plant Pathol. 106:623, 2000. (2) R. R. Martin. Can. J. Plant. Pathol. 6:264, 1984. (3) A. F. Murant. Raspberry Bushy Dwarf. Page 229 in: Virus Diseases of Small Fruits. R. H. Converse, ed. U.S. Dep. Agric. Agric. Handb. 631, 1987. (4) B. Strik and R. R. Martin. Plant Dis. 87:294, 2003.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.38.1.120-124.2000 ◽

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.

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A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope

Blood ◽

10.1182/blood.v77.9.1964.1964 ◽

1991 ◽

Vol 77 (9) ◽

pp. 1964-1969 ◽

Cited By ~ 22

Author(s):

F Flug ◽

R Espinola ◽

LX Liu ◽

C SinQuee ◽

R DaRosso ◽

...

Keyword(s):

Amino Acid ◽

Polyclonal Antibodies ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Adenosine Diphosphate ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Polymorphism ◽

Glycoprotein Iiia ◽

Polymerase Chain ◽

Induced Aggregation

Abstract We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105–129 and 452- 428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67- Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.

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

Blood ◽

10.1182/blood.v65.2.496.496 ◽

1985 ◽

Vol 65 (2) ◽

pp. 496-500 ◽

Cited By ~ 16

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.

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Characterization, purification and synthesis of spore-coat protein in Bacillus megaterium KM

Biochemical Journal ◽

10.1042/bj2020231 ◽

1982 ◽

Vol 202 (1) ◽

pp. 231-241 ◽

Cited By ~ 10

Author(s):

G S A B Stewart ◽

D J Ellar

Keyword(s):

Coat Protein ◽

Bacillus Megaterium ◽

Early Stage ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Stage Iv ◽

Spore Coat ◽

Methionine Residue ◽

Protein Incorporation ◽

Spore Coat Protein

The spore-coat fraction from Bacillus megaterium KM, when prepared by extraction of lysozyme-digested integuments with SDS (sodium dodecyl sulphate) and urea, contains three N-terminal residues and a major component of apparent mol.wt. 17500. Electron microscopy of this fraction shows it to consist of an ordered multilamellar structure similar to that which forms the coat region of intact spores. The 17500-dalton protein, which has been purified to homogeneity, has an N-terminal methionine residue, has high contents of glycine, proline, cysteine and acidic amino acids and readily polymerized even in the presence of thiol-reducing agents. It is first synthesized between late Stage IV and early Stage V, which correlates with the morphological appearance of spore coat. Before Stage VI the 17500-dalton protein is extractable from sporangia by SDS in the absence of thiol-reducing reagents. Between Stage VI and release of mature spores the protein becomes resistant to extraction by SDS unless it is supplemented by a thiol-reducing reagent. In addition to that of the spore-coat protein, the timing of synthesis of all the integument proteins was analysed by SDS/polyacrylamide-gel electrophoresis and non-equilibrium pH-gradient electrophoresis. Several integument proteins are conservatively synthesized from as early as 1h after the end of exponential growth (t1), which may reflect protein incorporation into the spore outer membrane.

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