Determination of pools of tricarboxylic acid cycle and related acids in bacteria.

Methods for sampling, extracting, and quantitating the metabolic pools of organic acids from bacteria have been developed. The concentration of these metabolites was determined by a new gas chromatographic method that can quantitatively determine the levels of lactate, pyruvate, fumarate, succinate, malate, alpha-ketoglutarate, and citrate. Values obtained were confirmed by fluorimetric analyses of five of the individual acids. In Escherichia coli, pools range from about 1 to 5 mumol/g of dry weight, with a variation in replicate samples of 5 to 15%. Under similar conditions, these pools in Bacillus licheniformis are in the same range, although the pyruvic acid pool is significantly larger.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. II. IgG antibodies contain VH genes from a single VH family and VL genes from at least four VL families

To define the V gene family repertoire of human IgG anti-Haemophilus influenzae type b polysaccharide antibodies, we purified six IgG1 and nine IgG2 anti-Hib-PS antibodies to monoclonality from immune serum of six individuals and performed N-terminal amino acid sequence analysis. Of the 15 clonal antibodies we examined, all H chain V regions were of the VHIII family. In contrast, the L chains of these antibodies were clearly from at least four different VL families; VKI, VKII, VKIII, and V lambda. Interestingly. VL family expression correlated with the cross-reactivity of these antibodies to the capsular carbohydrate of Escherichia coli K100. VKII antibodies did not cross-react, whereas antibodies expressing V lambda, VKI, or VKIII generally cross-reacted. We conclude that L chain V regions are very important contributors to the limited heterogeneity in this antibody repertoire.     

Activation of the Mitogen-activated Protein Kinase,Slt2p,at Bud Tips Blocks a Late Stage of Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae

Inheritance of the endoplasmic reticulum (ER) requires Ptc1p, a type 2C protein phosphatase of Saccharomyces cerevisiae. Genetic analysis indicates that Ptc1p is needed to inactivate the cell wall integrity (CWI) MAP kinase, Slt2p. Here we show that under normal growth conditions, Ptc1p inactivates Slt2p just as ER tubules begin to spread from the bud tip along the cortex. In ptc1Δ cells, the propagation of cortical ER from the bud tip to the periphery of the bud is delayed by hyperactivation of Slt2p. The pool of Slt2p that controls ER inheritance requires the CWI pathway scaffold, Spa2p, for its retention at the bud tip, and a mutation within Slt2p that prevents its association with the bud tip blocks its role in ER inheritance. These results imply that Slt2p inhibits a late step in ER inheritance by phosphorylating a target at the tip of daughter cells. The PI4P5-kinase, Mss4p, is an upstream activator of this pool of Slt2p. Ptc1p-dependant inactivation of Slt2p is also needed for mitochondrial inheritance; however, in this case, the relevant pool of Slt2p is not at the bud tip.     

Cross-linking of collagen in the X-linked Ehlers-Danlos Type V.

The activity and antigenicity of the collagen crosslinking enzyme, lysyl oxidase, and the proportions of reducible crosslink in skin biopsies from Ehlers-Danlos Type V subjects were equivalent to those of control skin. These results reveal that both the potential for crosslinking, and the ability to form reducible crosslinks is present in Ehlers-Danlos syndrome Type V subjects, clearly demonstrating that the defect in this disorder is not due to a defective crosslinking mechanism.