Southern elephant seal,Mirounga leonina: composition of milk during lactation

An analysis of milk constituents during various stages of lactation in the southern elephant seal Mirounga leonina was carried out. Forty-six milk samples were taken from 30 females throughout lactation during 1985, 1987, 1990 and 1991 on Stranger Point, King George Island, South Shetland Islands. Total nitrogen (TN), non-protein nitrogen (NPN), sugar, fat, ash and water were measured, and from some of these data true protein and energy content were calculated. The results showed a high degree of variation in water and fat concentrations among samples at different stages of lactation. During the first 20 days the fat content of milk increased from about 12 to approximately 52%, while water content fell from 70 to 33%. The composition of milk changes rapidly during the first days post-partum. Protein, minerals and sugar appear to remain stable after the fourth day of lactation. Milk samples contain significant levels of sugars; thin layer chromatography indicates the presence of lactose and glucose together with other unidentified components. There is evidence of a striking change in composition of the milk in the later part of lactation; the progressive increase in the fat:water ratio is abruptly reversed just prior to weaning.     

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

A structural and evolutionary analysis of a dispersed repetitive sequence

A family of dispersed repetitive sequences (Hch1) which is present in the genome of the wild barley Hordeum chilense was studied in detail. Hch1 sequences are found both as part of short tandem arrays and dispersed throughout the H. chilense chromosomes. Subcloning of sections of the sequence reveals that it is composed of unrelated classes of sequences which can also be found separately in other genomic locations. Analysis of these sequences in the genomes of wheat and two other wild barley species strongly suggests that specific amplifications and arrangements of the repeated sequences have taken place during speciation. Nucleotide sequence analysis fails to detect, in their entirity, the features shown by plant transposons.     

CD studies of peptides that mimic the four helicoidal sequences of the uteroglobin monomer

Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P2₁ and C222₁) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P2₁). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process.     

Methane production from acetamide in an upflow anaerobic sludge-blanket reactor based on a synergistic association between an aerobic rod and methanogens

Acetamide degradation was investigated in a bench-scale upflow anaerobic sludge-blanket (UASB) reactor, successively fed with acetamide, acetate and acetamide, over a period of 343 days, at different hydraulic retention times (t _HR). The reactor was seeded with the sludge previously described [Guyot et al. (1994) Appl Microbiol Biotechnol, 42:452-456], in which methanogenesis from acetamide was performed through a synergistic relationship between an acetamide-degrading, aerobic rod and methanogens. When the reactor was fed acetamide, the chemical oxygen demand (COD) removal efficiency was 86% at volumetric loads less than 1.18 kg COD m^–3 day ^–1. At higher volumetric loads, the efficiency decreased markedly, e.g. 50.9% at a volumetric organic load of 3.39 kg COD m^–3 day^–1 (1 day t _HR) with an accumulation of both acetamide and acetate. The same reactor, when fed with acetate at t _HR 1 day, reached a high COD removal (99%). Evidence of the inhibition of acetate degradation by acetamide is presented. After a long period (135 days) without feeding the reactor with acetamide, the sludge reactor was still capable of degrading acetamide when this substrate was supplied again. It seems that the synergistic degradation of acetamide by aerobes and methanogens present in the UASB reactor sludge is stable over a long period (343 days), in spite of limiting concentrations of dissolved oxygen in the feed.     

Organosolv pulping of wheat straw by use of phenol

A central composite design was used to investigate the influence of the cooking conditions (time, temperature and phenol concentration) for wheat straw with phenol-water mixtures on the properties of the pulp obtained (yield and holocellulose, -cellulose, lignin and ethanol-benzene extractable contents) and the pH of the resulting wastewater. A second-order polynomial model consisting of three independent process variables was found to accurately describe the organosolv pulping of wheat straw. The equations derived predict the yield, the holocellulose, -cellulose, lignin and ethanol-benzene extractable contents of the pulp, and the pH of the wastewater with multiple-R, R² and adjusted-R² high values. The process variables must be set at low variables in order to ensure a high yield and pH. Conversely, if high holocellulose and -cellulose contents, and low lignin and ethanol-benzene extractable contents are desired, then a high temperature (200°C), long cooking time (120 min), and intermediate phenol concentration (65%) must be used.     

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from aKlebsiella pneumoniae mutant strain

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA⁺ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.     

Phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase has been purified to apparent homogeneity by a process involving the following steps: solubilization from microsomes and chromatography on Affi-Gel Blue, phosphocellulose, Bio-Gel A 1.5m, and agarose-hexane-ATP. The apparent M_r of the purified enzyme as judged by gel-filtration chromatography is 205,000 and by sodium dodecyl sulfate-gel electrophoresis is 105,000. Immunoprecipitation of homogeneous reductase phosphorylated by reductase kinase and [γ-³²P]ATP produces a unique band containing ³²P bound to protein which migrates at the same R_f as the reductase subunit. Incubation of ³²P-labeled HMG-CoA reductase with reductase phosphatase results in a time-dependent loss of protein-bound ³²P radioactivity, as well as an increase in enzymic activity. Reductase kinase, when incubated with ATP, undergoes autophosphorylation, and a simultaneous increase in its enzymatic activity is observed. Tryptic treatment of immunoprecipitated, ³²P-labeled HMG-CoA reductase phosphorylated with reductase kinase produces only one ³²P-labeled phosphopeptide with the same R_f as one of the two tryptic phosphopeptides that have been reported in a previous paper. The possible existence of a second microsomal reductase kinase is discussed.     

Three new species of penicillium

Three new species of microfungi belonging to the genus Penicillium Link ex Fries are described and illustrated. All but one have been isolated from the atmosphere of las Palmas, capital city of the island of Gran Canaria (Canary Islands, Spain). They clearly differ from all species of the genus described so far and are, therefore, described and proposed as new species: Penicillium hispanicum sp. nov., Penicillium grancanariae sp. nov., and Penicillium palmensis sp. nov.