Effect of metabolic alkalosis on respiratory function in patients with chronic obstructive lung disease.

Eleven instances of a mixed acid-base disorder consisting of chronic respiratory acidosis and metabolic alkalosis were recognized in eight patients with chronic obstructive lung disease and carbon dioxide retention. Correction of the metabolic alkalosis led to substantial improvement in blood gas values and clinical symptoms. Patients with mixed chronic respiratory acidosis and metabolic alkalosis constitute a common subgroup of patients with chronic obstructive lung disease and carbon dioxide retention; these patients benefit from correction of the metabolic alkalosis.     

Stimulation of glucose transport in rat adipocytes by insulin, adenosine, nicotinic acid and hydrogen peroxide. Role of adenosine 3′:5′-cyclic monophosphate

Glucose transport into adipocytes of the rat was measured by monitoring the conversion of [1-(14)C]glucose into (14)CO(2). Glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent that rapidly reoxidizes NADPH. Under these conditions, the observed rate of glucose disappearance from the incubation medium was about 20% higher than the rate of conversion of the C-1 of glucose into (14)CO(2). Apparent rates of glucose transport were significantly increased by insulin, H(2)O(2), adenosine and nicotinic acid. Stimulation of the apparent rate of glucose transport by insulin was dependent on adipocyte concentration, the hormone being most effective at relatively high cell concentrations. Adenosine and nicotinic acid further enhanced the maximum stimulation of glucose transport by insulin. Potentiation of insulin action by adenosine was more pronounced at lower cell concentrations. At relatively high cell concentrations the stimulatory action of insulin was markedly decreased by adenosine deaminase. Stimulation of apparent rates of glucose transport by the compounds noted above were antagonized by agents that increased intracellular cyclic AMP concentrations (theophylline and isoprenaline) and by dibutyryl cyclic AMP. Intracellular concentrations of cyclic AMP were significantly lowered when adipocytes were incubated with insulin, H(2)O(2), adenosine or nicotinic acid. These effects were observed under basal conditions or when intracellular cyclic AMP concentrations were elevated by theophylline or isoprenaline. On the basis of the above data, we suggest that insulin, H(2)O(2), adenosine and nicotinic acid may all stimulate glucose transport in rat adipocytes by lowering the intracellular cyclic AMP concentration. These data therefore support the hypothesis that cyclic AMP inhibits glucose transport in rat adipocytes.     

Role of the mitochondrial anion transporters in the regulation of ammoniagenesis in renal cortex mitochondria of the rabbit and rat.

The purpose of this study was to investigate factors which may regulate ammoniagenesis in the kidney cortex. Emphasis was placed on the segment of the pathway by which the carbons derived from glutamine must exit from the mitochondrion. These pathways were compared in the rat with high rates of ammoniagenesis and the rabbit which has a low rate of ammoniagenesis. The dicarboxylate transporter, which is essential for ammoniagenesis, has a maximum velocity which was much lower in the rabbit. The malate concentration required for half-maximal rates of transport was 14 nmol/mg mitochondrial protein and similar in both species. There was no effect of chronic metabolic acidosis on dicarboxylate transporter activity. The tricarboxylate transporter activity with phosphoenol pyruvate as substrate also had a low activity in the rabbit kidney-cortex mitochondria. The maximum velocity of phosphate dependent glutaminase, glutamate dehydrogenase and phosphoenolpyruvate carboxykinase were all much greater than the maximal rate of ammoniagenesis observed in vivo in the rabbit. Therefore, the low rates of ammoniagenesis and the failure to adapt to acidosis in the rabbit are best explained by factors influencing the dicarboxylate transporter.     

Invasive Haemophilus influenzae type b infections in vaccinated and unvaccinated children in Canada, 2001–2003

Background

Although vaccination of infants against Haemophilus influenzae type b (Hib) invasive infections is effective and has been routinely available in Canada since 1992, cases of the disease continue to occur. We were interested in determining whether recent cases of Hib infection reflected progressive loss of protection with time since vaccination, increasing nonacceptance of vaccination or a deleterious effect of coadministration of recently introduced vaccines such as those for pneumococcal and meningococcal conjugates and hepatitis B. We report on the causes of Hib infections among vaccinated and unvaccinated children between 2001 and 2003 in Canada.

Methods

Through our established network of 12 pediatric tertiary care hospitals we actively searched for cases in each centre by reviewing daily admissions and laboratory reports, visiting the wards and checking discharge diagnosis codes. Culture-confirmed cases were summarized by nurse monitors using a standardized reporting system.

Results

We identified 29 cases during the 3 years: 16 in 2001, 10 in 2002 and 3 in 2003. Half of the 29 patients had meningitis. Hib infection was more common among children less than 6 months of age (11 cases) and in boys (20 cases). Two deaths occurred (7% case-fatality ratio). A total of 20 children had received no or incomplete primary vaccination because of parental refusal (7 cases), because they were too young to have completed the primary series (11 cases, including 1 in which parental refusal was also a factor) or because of delays in completing the primary series (2 cases); the vaccination history was uncertain in the remaining case. Infection despite primary vaccination occurred in 9 children: 2 previously healthy children and 7 who were immunocompromised or who had a predisposing condition. None of the cases identified in 2003 involved children who had received any of the newly introduced vaccines.

Interpretation

Invasive Hib infections remain rare in Canada, with most cases occurring in children too young to have completed the primary series. Protection after vaccination appears to extend into later childhood and does not appear to be diminished by coadministration of newer infant vaccines.Until recently Haemophilus influenzae type b (Hib) was a leading cause of meningitis, epiglottitis and other invasive infections in children, affecting about 1 child in 250 by 5 years of age.¹ The risk of infection was highest among children 6–24 months of age. Antibodies directed against the Hib capsular polysaccharide (polyribosyl ribitol phosphate, PRP) form the basis of protection. PRP protein conjugate vaccines that elicit anti-PRP responses in young infants have been used in Canada since 1992. Doses are recommended at 2, 4 and 6 months of age to establish protection and at 18 months to reinforce it. Since 1995 all provinces have used the same Hib vaccine (a PRP–tetanus protein conjugate [PRP-T], produced by Aventis Pasteur), in combination products based on whole-cell pertussis vaccines (from 1995 to 1997) or acellular pertussis vaccines (1998 to the present).Invasive Hib infections have been monitored since 1992 by a network of Canadian pediatric hospitals known as the Immunization Monitoring Program, Active (IMPACT).² In 1985, before the first Hib vaccine was licensed, 485 invasive Hib cases were seen at 10 centres (those participating in IMPACT when the “look-back” was done).³ Case totals fell progressively as better vaccines became available.^3,4,5 In 2000, only 4 cases were recorded by the IMPACT centres (which by then numbered 12), 99% fewer than in 1985.⁶ Continuing surveillance is important to assess the effectiveness of the current schedule and vaccine. Because Hib vaccination is relatively new, the question of duration of protection remains open. Resurgence of Hib disease occurred recently in the United Kingdom,⁷ prompting addition of a booster dose to the vaccination schedule (as in Canada). Other questions of relevance are whether nonacceptance of Hib vaccine is influencing case totals and whether coadministration of newer vaccines, such as those for pneumococcal and meningococcal group C conjugates and hepatitis B, is adversely affecting Hib responses. A reduced response is most likely to occur when infants are given conjugate vaccines containing the same carrier protein,⁸ which is not the case with PRP-T and pneumococcal conjugate vaccines; however, their compatibility has not been formally demonstrated to date. In this report we present details of cases encountered by IMPACT in the period 2001 to 2003.     

ADVENTIVE EMBRYONY IN TISSUE CULTURES OF THE WILD CARROT,DAUCUS CAROTA

Cultures of callus tissue derived from roots, petioles, or umbellet peduncles of the wild carrot have been observed to form large numbers of adventive embryos which closely resemble ovular embryos of the same species. In the presence of cocoanut milk, which is not otherwise required in the nutrient medium, these embryos germinate and produce plants which may be normal in all respects. The sequence of forms in embryogenesis has been traced back to embryos of fewer than 10 cells, and the pattern of development has been found in many cases to correspond closely to that of ovular embryos, especially with respect to the early filamentous stages. The accumulation of granular starch in both the parenchymatous cells of the callus and in the embryos at certain stages of their development is a marked characteristic of differentiating callus. Investigation of the nutrient requirements of the callus tissue undergoing differentiation of embryos reveals that a wide latitude is possible in the composition of the basal medium, consisting of mineral salts, vitamins, and sucrose. Embryos form readily in tissue which has been extensively subcultured on a simple defined medium which consists of the basal medium plus adenine and 2,4-D. Tissues cultured on cocoanut milk and 2,4-D, or kinetin and 2,4-D, are more heterogeneous in composition and may contain, in addition to embryos, idioblastic tracheids, anthocyanin-pigmented cells, and complex vascularized nodules. Chemical control of alternative morphogenetic pathways, and of embryogenesis itself, is partially demonstrable.     

Degradation of mistargeted OEE33 in the chloroplast stroma

OEE33, a component of the oxygen-evolving enzyme in chloroplasts, normally resides in the thylakoid lumen. In an attempt to study the fate of mistargeted proteins in chloroplasts, we substituted the bipartite transit peptide of OEE33 with that of CAB7, an integral thylakoid-membrane protein. As a result, when imported into isolated chloroplasts, the chimeric protein was targeted to the stroma instead of the thylakoid lumen. Whereas the wild-type OEE33 was totally stable for at least 2 h, the chimeric protein was rapidly degraded, with a half-life of 60 min. Degradation of the chimeric protein was stimulated by ATP supplementation. Degradation could also be observed in lysed chloroplasts, in an ATP-stimulated manner. When lysates were fractionated, the proteolytic activity was found to be associated mainly with the stromal fraction. This activity was very effectively inhibited by all tested inhibitors of serine proteases. Western blot analysis demonstrated that the stromal fraction active in degrading the chimeric OEE33 contains ClpC and ClpP, homologues of the regulatory and proteolytic subunits, respectively, of the bacterial, ATP-dependent, serine-type Clp protease.     

The role of the cytoplasmic redox potential in the control of fatty acid synthesis from glucose, pyruvate and lactate in white adipose tissue

The metabolism of lactate, pyruvate and glucose was studied in epididymal adipose tissue of starved, normally fed and starved-re-fed rats. Lactate conversion into fatty acid occurred at an appreciable rate only in the adipocyte of starved-re-fed animals. NNN'N'-Tetramethyl-p-phenylenediamine, an agent that transports reducing power from the cytoplasm to the mitochondria, caused large increments of fatty acid synthesis from lactate and a smaller one from glucose but a decrease in that from pyruvate. Glucose (1.0mm) increased fatty acid synthesis from lactate 4.3-fold but only 1.67-fold from pyruvate in adipocytes from normally fed animals. 2-Deoxyglucose decreased fatty acid synthesis from lactate to a greater degree (threefold) compared to that from pyruvate in adipocytes from starved-re-fed animals. l-Glycerol 3-phosphate contents were approximately equal in epididymal fat-pads, incubated in the presence of lactate or pyruvate, from normally fed animals, whereas the addition of 1mm-glucose resulted in a tenfold increase in l-glycerol 3-phosphate content only in the presence of lactate. The l-glycerol 3-phosphate content was tenfold higher in adipose tissue from starved-re-fed animals incubated in the presence of lactate than in the presence of pyruvate. 2-Deoxyglucose caused these values to be slightly lowered in the presence of lactate. We suggest that lactate metabolism is limited by the rate of NADH removal from the cytoplasm. In the starved-re-fed state, this occurs by reduction of dihydroxyacetone phosphate formed from glycogen to produce l-glycerol 3-phosphate, thus permitting lactate conversion into fatty acid. When glucose is the substrate, and rates of transport are not limiting, the rate of removal of cytoplasmic NADH limits glucose conversion into fatty acid.     

The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C‐terminal cysteine of the CXXC motif

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N‐terminal cysteine interacts with substrates, whereas the C‐terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C⁸⁶P⁸⁷D⁸⁸C⁸⁹ catalytic motif. In vitro, SdbA single cysteine variants at the N or C‐terminal position (SdbA_C86P and SdbA_C89A) were active but displayed different susceptibility to oxidation, and N‐terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N‐terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C‐terminal cysteine alone (in the SdbA_C86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C‐terminal cysteine.     

Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasmid pTiBo542.

Agrobacterium tumefaciens strains carrying pTiBo542 induce large, fast-appearing tumors and have an unusually wide host range. A clone bank was made from this 250-kilobase plasmid in a wide-host-range vector, and restriction maps were determined for BamHI and SalI. The virulence genes, transferred DNA genes, plasmid incompatibility region, and a region that inhibits growth of certain A. tumefaciens strains were localized. The six virulence genes and two tms genes were highly homologous to the genes of pTiA6, but the tmr gene was not. Mutations in each of the six vir loci of pTiA6 were complemented by clones from the vir region of pTiBo542.     

Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein

A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.