Growth and hematologic development of the patas monkey (Erythrocebus patas) to one year of age.

Data from 338 blood samples of 31 patas monkeys is presented to show the normal hematolgoic development from birth to one year of age, and is compared to 120 samples from normal adults. Marked changes in hematocrit, hemoglobin, red cell numbers, and leukocyte distribution which occurred during the first month were followed by a slow progression to adult values. Body weight data is also presented which shows a linear growth rate during the first year of life.     

Structure,function and applications of carbonic anhydrase isozymes

The carbonic anhydrases enzymes (CAs, EC 4.2.1.1) are zinc containing metalloproteins, which efficiently catalyse the reversible conversion of carbon dioxide to bicarbonate and release proton. These enzymes are essentially important for biological system and play several important physiological and patho-physiological functions. There are 16 different alpha-carbonic anhydrase isoforms studied, differing widely in their cellular localization and biophysical properties. The catalytic domains of all CAs possess a conserved tertiary structure fold, with predominately β-strands. We performed an extensive analysis of all 16 mammalian CAs for its structure and function in order to establish a structure–function relationship. CAs have been a potential therapeutic target for many diseases. Sulfonamides are considered as a strong and specific inhibitor of CA, and are being used as diuretics, anti-glaucoma, anti-epileptic, anti-ulcer agents. Currently CA inhibitors are widely used as a drug for the treatment of neurological disorders, anti-glaucoma drugs, anti-cancer, or anti-obesity agents. Here we tried to emphasize how CAs can be used for drug discovery, design and screening. Furthermore, we discussed the role of CA in carbon capture, carbon sensor and metabolon. We hope this review provide many useful information on structure, function, mechanism, and applications of CAs in various discipline.     

Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes.

PCR of cDNA produced from patient fibroblasts allowed us to determine the paternal mutation in the first patient reported with beta-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G-->T transversion 1,881 bp downstream of the ATG translation initiation codon destroys an MboII restriction site and converts Trp627 to Cys (W627C). Digestion of genomic DNA PCR fragments with MboII indicated that the patient and the father were heterozygous for this missense mutation in exon 12. Failure to find cDNAs from patient RNA which did not contain this mutation suggested that the maternal mutation leads to greatly reduced synthesis or reduced stability of mRNA from the mutant allele. In order to identify the maternal mutation, it was necessary to analyze genomic sequences. This approach was complicated by the finding of multiple unprocessed pseudogenes and/or closely related genes. Using PCR with a panel of human/rodent hybrid cell lines, we found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed us to amplify and characterize genomic sequences for the true beta-glucuronidase gene despite this background of related sequences. The patient proved to be heterozygous for a second mutation, in which a C-->T transition introduces a termination codon (R356STOP) in exon 7. The mother was also heterozygous for this mutation. Expression of a cDNA containing the maternal mutation produced no enzyme activity, as expected. Expression of the paternal mutation in COS-7 cells produced a surprisingly high (65% of control) level of activity. However, activity was 13% of control in transiently transfected murine MPS VII cells. The level of activity of this mutant allele appears to correlate with the level of overexpression, suggesting that high concentrations of mutant monomers can drive the folding and tetramerization of mutant enzyme to produce an active and stable enzyme.     

Development of a biofilter for the removal of methane from coal mine ventilation atmospheres

Several strains of methane-oxidizing bacteria were isolated and studied to determine their physiological suitability for removal of methane in coal mine atmospheres. One strain, Methylomonas fodinarum ACM 3268, was selected as the most suitable culture for use in the development of a continuous biofilter to be used as a ventilation air scrubber. The experimental biofilter utilising a biofilm of M. fodinarum was shown to reduce methane levels substantially provided the residence times were sufficiently long. In the range 0.25–1.0% methane in air, commonly experienced in coal mine atmospheres, more than 70% of the methane was removed with a residence time of 15 min, with a 90% reduction at 20 min. Even at a residence time of 5 min approximately 20% of the methane in air was removed. Equal quantities of O₂ are consumed during the bacterial oxidation of methane and 1% methane is converted to 0.7% CO₂. Scale-up and alternative biofilter packings are likely to reduce the residence times in the biofilter.     

Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation

Aspergillus foetidus ACM 3996 (=FRR 3558) and three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846) were compared for the production of citric acid from pineapple peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4g of citric acid per 100g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30°C, an unadjusted initial pH of 3.4, a particle size of 2mm and 5ppm Fe²⁺. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.     

Comparative kinetics of phosphomannosyl receptor-mediated pinocytosis of fibroblast secretion acid hydrolases and glycopeptides prepared from them

In a previous report we demonstrated that phosphorylated oligosaccharides isolated from acid hydrolases were subject to pinocytosis by phosphomannosyl receptors present on the cell surface of human fibroblasts [9]. However, limiting quantities of oligosaccharides precluded detailed comparison of the kinetics of pinocytosis of these phosphorylated oligosaccharides to those of the acid hydrolases from which they were derived. In this report we present studies comparing the kinetics of pinocytosis of acid hydrolases from NH4Cl-induced fibroblast secretions with those of concanavalin A-binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125I-labeled glycopeptides was linear for at least 3 hr, saturable, inhibited competitively by mannose 6-phosphate, and destroyed by prior treatment of the ligand with alkaline phosphatase. The inhibition constants of excess unlabeled glycopeptide for the uptake of 125I-labeled glycopeptides (Ki of 1.5 X 10(-6) M) and for the uptake of secretion acid hydrolases (Ki of 2.2 X 10(-6) M) were remarkably similar. Furthermore, the Ki for mannose 6-phosphate inhibition of pinocytosis of glycopeptide uptake (3 X 10(-5) M) compares closely to that previously determined for the pinocytosis of intact "high-uptake" acid hydrolases (3-6 X 10(-5) M). "High-uptake" fractions of both ligands were prepared and quantified by affinity chromatography on immobilized phosphomannosyl receptors purified from bovine liver. Only 10% of the concanavalin A-binding glycopeptides bound to the immobilized phosphomannosyl receptors, while 80% of the acid hydrolases from which they were prepared bound and were eluted with 10 mM mannose 6-phosphate. However, the fraction of each type of ligand that binds to the immobilized phosphomannosyl receptors accounts for all the uptake activity of that ligand.     

Histamine-induced constriction of canine peripheral lung: an airway or tissue response?

We compared the histamine responsiveness of peripheral airways (less than 6.0 mm diam) and parenchymal tissues in eight anesthetized paralyzed open-chest mongrel dogs. We measured pressure in a peripheral bronchus by using an antegrade wedged catheter and pressure in the alveolar region subtended by the wedged bronchus by using an alveolar capsule. Sinusoidal volume oscillations at a frequency of 0.5 Hz were delivered by a linear motor pump into the segment through the wedged catheter. We calculated the resistance of the segment (Rseg) and partitioned Rseg into tissue viscance (i.e., proportional to the resistive pressure drop between the alveolus and the pleura) and peripheral airway resistance. Measurements were taken under baseline conditions and after delivery of increasing concentrations of aerosolized histamine (0.1 micrograms/ml to 100.0 mg/ml) into the segment. We found that the histamine responsiveness of the peripheral airways and lung tissues varied markedly within a given dog. In four of eight dogs the airways were more responsive to histamine, in three of eight the tissues were more responsive, and in one of eight the response was equivalent at the two sites. We conclude that in a given animal, there is marked heterogeneity in the histamine responsiveness of the peripheral airways and parenchymal tissues and that either may dominate responsiveness in the peripheral lung.     

Binding of phosphorylated oligosaccharides to immobilized phosphomannosyl receptors

We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had properties similar to the previously reported binding of enzyme to receptors on cell surfaces and isolated membranes. In order to determine the structural features of the phosphomannosyl recognition marker required for receptor recognition, we covalently coupled purified receptor to an agarose gel bead support for affinity chromatography of phosphorylated, high mannose-type oligosaccharides isolated from fibroblast secretions radiolabeled with [2-3H]mannose. Neutral oligosaccharides and oligosaccharides containing one or two phosphates in phosphodiester linkage were not retained by the receptor column. By contrast, oligosaccharides bearing one phosphomonoester moiety were retarded on the column; those bearing two phosphomonoesters were bound to the column and were eluted with 10 mM mannose 6-phosphate. The binding of the oligosaccharides to the immobilized receptor correlates with their ability to be pinocytosed by fibroblasts and shows that the preferred recognition marker for the phosphomannosyl-enzyme receptor is a high mannose-type oligosaccharide chain bearing two uncovered phosphomannosyl groups.     

Membrane-associated carbonic anhydrase from rat lung. Purification, characterization, tissue distribution, and comparison with carbonic anhydrase IVs of other mammals.

Carbonic anhydrase (CA) IV was purified to homogeneity from rat lung microsomal and plasma membranes. The single N-terminal amino acid sequence showed 55% similarity to that reported for human CA IV. A monospecific antibody to the 39-kDa rat enzyme that cross-reacts on Western blots with CA IVs from other mammalian species was produced in rabbits. Digestion of rat lung enzyme with endoglycosidase (peptide-N-glycosidase F) reduced the Mr to 36,000, suggesting that rat CA contains one N-linked oligosaccharide chain. All of eight additional mammalian CA IVs that were examined also contained oligosaccharide chains, as evidenced by reduction in Mr from 52,000 (cow, sheep, and rabbit), 42,000 (pig, guinea pig, and dog), and 39,000 (mouse and hamster) to 36,000 after treatment of the respective lung microsomal membranes with peptide-N-glycosidase F. The 36-kDa human enzyme showed no change in molecular mass with this treatment. Thus, the human CA IV is the exceptional one in lacking carbohydrate. Rat lung CA IV was found to be relatively resistant to sodium dodecyl sulfate and to be anchored to membranes by a phosphatidylinositol-glycan linkage; both properties were found to be shared by other mammalian CA IVs. Western blot analysis indicated distribution of CA IV in rat tissues other than kidney and lung where it was previously known to be present. CA IV was particularly abundant in rat brain, muscle, heart, and liver, all locations where the CA IV enzyme was not known to be present previously. None was detected in rat skin or spleen.