The structure of the neutral polysaccharide gum secreted by Rhizobium strain cb744

A previous investigation of the structure of the extracellular polysaccharide gum from the nitrogen-fixing Rhizobium strain cb744 (a member of the slow-growing Cowpea group) indicated that there were two β-(1→4)-linked d-glucopyranosyl residues for each α-(1→4)-linked d-mannopyranosyl residue, and that each mannose was substituted at O-6 by a β-d-galactopyranosyl residue having 71% of the galactose present as 4-O-methylgalactose. The present study shows that, although the gum appeared to have a simple tetrasaccharide repeating unit, it is composed of two closely associated components. One is a (1→4)-linked α-d-mannan substituted at each O-6 by a β-d-galactopyranosyl residue (71% 4-O-methylated). The second component is a (1→4)-linked β-d-glucan. The existence of the two polysaccharides was established by separation of the β-d-galactosidase-treated gum on a column of concanavalin A-Sepharose 4B. The d configurations were determined and the anomeric attribution of the linkages confirmed by the use of enzymes. The interaction between the two gum components is discussed.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Cervical sympathetic and phrenic nerve responses to progressive brain hypoxia

To determine if depression of central respiratory output during progressive brain hypoxia (PBH) can be generalized to other brain stem outputs, we examined the effect of PBH on the tonic (tSCS) and inspiratory-synchronous (iSCS) components of preganglionic superior cervical sympathetic (SCS) nerve activity. Peak phrenic and SCS activity were measured in nine anesthetized, paralyzed, peripherally chemodenervated, vagotomized cats. PBH was produced by inhalation of 0.5% CO in 40% O2 while blood pressure and end-tidal CO2 were maintained constant. A progressive reduction in arterial O2 content from 14.3 +/- 0.6 to 4.5 +/- 0.3 vol% caused a 79 +/- 7% depression of peak phrenic activity and an 84 +/- 10% reduction of iSCS activity, but tSCS activity increased 42 +/- 21%. During CO2 rebreathing, iSCS activity increased in parallel with peak phrenic activity while tSCS activity was unchanged. The slopes of the CO2 responses of both phrenic (6.3 +/- 1.2%max/mmHg) and iSCS (4.6 +/- 0.8%max/mmHg) activity were unaffected by PBH. In four of nine hypocapnic and three of nine hypoxic studies, inspiratory activity in the SCS nerve was observed even after completely silencing the phrenic neurogram.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane Skeletal Proteins and Their Integral Membrane Protein Anchors are Targets for Tyrosine and Threonine Kinases in Euglena

ABSTRACT. Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [³²P]-orthophosphate or γ-[³²P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.     

Antitumor effects of an antibody-carboxypeptidase g2 conjugate in combination with a benzoic acid mustard prodrug

The F(ab’)₂ fragment of the antitumor monoclonal antibody, A5B7, was covalently linked to the bacterial enzyme carboxypeptidase G2 (CPG2). The resulting conjugate was used in combination with a prodrug of a benzoic acid mustard alkylating agent to treat human colon tumor xenografts in a two-step targeting strategy, antibody-directed enzyme produrug therapy (ADEPT). The prodrug, 4-[(2-chloroethyl) (2-mesyloxyethyl) amino]-benzoyl-l-glutamic acid is rapidly converted by CPG2 to a drug that is at least 15x more toxic in vitro against LS174T colorectal tumor cells than the prodrug. Optimal tumor/ blood ratios of the A5B7-CPG2 were achieved 72 h after administration of the conjugate to athymic mice bearing established LS174T tumor xenografts. Significant antitumor activity was seen in LS174T tumor-bearing mice treated with the conjugate followed 3 d later by the prodrug. In contrast, prodrug, conjugate, or active drug alone did not result in any antitumor activity in this tumor model. These studies demonstrate the advantage of a two-step ADEPT system for the treatment of colorectal cancer.