Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol

Commercial cellulase enzymes have been used in the food, detergent, and textile industries, and are potentially effective for processing biomass feedstocks. A survey was undertaken to identify major manufacturers/distributors of cellulases in the USA and to evaluate 13 representative commercial preparations for enzyme activity, protein concentration, and chemical composition. Samples were subjected to activity measurements using filter paper, carboxymethylcellulose, cellobiose, and p-nitrophenyl-β-d-glucopyranoside as substrates. To ascertain the microbial origin of the commercial preparations, Western blots utilizing monoclonal antibodies specific for Trichoderma reesei CBH I and Aspergillus niger β-d-glucosidase were developed. Eleven of the cellulases tested were of T. reesei or T. viride origin and two were from A. niger.     

Energy metabolism in relation to oxygen partial pressure in human skeletal muscle during exercise.

1. The intramuscular oxygen partial pressure (pO2) in human gastrocnemius muscle was monitored during exercise and compared with metabolite concentrations reflecting the energy and the redox state in the tissue. Ten normal subjects and ten patients with peripheral vascular occlusive disease were investigated. 2. In normal subjects the pO2 at the end of exercise was related to the intensity of the exercise, expressed as effect (J/s) per contraction. 3. In both patients and normal subject the pO2 was related to the [ATP]/[ADP] ratio, the [lactate/[pyruvate] ratio and the phosphocreatine concentration in the muscle tissue at rest and during exercise. 4. At each pO2 value, a lower [lactate/[pyruvate] ratio was found in the muscle tissue of the patients compared with that of normal subjects. This was interpreted as a beneficial effect of the higher oxidative-enzyme capacity in the muscle of the patients. 5. The results show the importance of pO2 for the regulation of the energy and the redox state of the tissue. During exercise the changes induced in pO2 and thus the energy state will stimulate the respiratory rate. This might be an important link in triggering the oxidative-enzyme capacity in response to physical training as well as in peripheral vascular occlusive disease.     

Decreased production ofpara-hydroxypenicillin V in penicillin V fermentations

Summary Penicillin V (phenoxymethyl penicillin) is produced by industrial strains ofPenicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain ofP. chrysogenum produces an undesirable penicillin byproduct,para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, viapara-hydroxylation of POAc and subsequent incorporation of thep-OH phenoxyacetic acid into the penicillin molecule. Most of thep-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level ofp-OH penicillin V produced by the control strain ranges up to 10–15% of the total penicillins produced. 3-Phenoxypropionic acid andp-bromophenylacetic acid partially inhibit the formation ofp-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels ofp-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which producep-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.     

The β-lactam antibiotics: past, present, and future

The discovery and development of the -lactam antibiotics are among the most powerful and successful achievements of modern science and technology. Since Fleming's accidental discovery of the penicillin-producing mold, seventy years of steady progress has followed, and today the -lactam group of compounds are the most successful example of natural product application and chemotherapy. Following on the heels of penicillin production by Penicillium chrysogenum came the discoveries of cephalosporin formation by Cephalosporium acremonium, cephamycin, clavam and carbapenem production by actinomycetes, and monocyclic -lactam production by actinomycetes and unicellular bacteria. Each one of these groups has yielded medically-useful products and has contributed to the reduction of pain and suffering of people throughout the world. Research on the microbiology, biochemistry, genetics and chemistry of these compounds have continued up to the present with major contributions being made by both individual and collaborative groups from industry and academia. The discovery of penicillin not only led to the era of the wonder drugs but provided the most important antibiotics available to medicine. Continued efforts have resulted in the improvement of these compounds with respect to potency, breadth of spectrum, activity against resistant pathogens, stability and pharmacokinetic properties. On the research front, major advances are being made on structural and regulatory biosynthetic genes and metabolic engineering of the pathways involved. New semisynthetic compounds especially those designed to combat resistance development are being examined in the clinic, and unusual non-antibiotic activities of these compounds are being pursued. Although seventy years of age, the -lactams are not yet ready for retirement.     

Preservation of rat skeletal muscle energy metabolism by illumination

Skeletal muscle viability is crucially dependent on the tissue levels of its high energy phosphates. In this study we investigated the effect of the preservation medium Perfadex and illumination with Singlet Oxygen Energy (SOE). Singlet oxygen can be produced photochemically by energy transfer from an excited photosensitizer. The energy emitted from singlet oxygen upon relaxation to its triplet state is captured as photons at 634 nm and is here referred to as SOE. Rat hind limb rectus femoris muscles were preserved for five hours at 22 degrees C in Perfadex, saline, SOE illuminated Perfadex or SOE illuminated saline. Extracts of the muscles were analysed by 31P NMR. Data were analysed using two-way analysis of variance and are given as mean values micromol/g dry weight) +/- SEM. The ATP concentration was higher (p = 0.006) in saline groups (4.52) compared with Perfadex groups (2.82). There was no statistically significant difference in PCr between the saline groups (1.25) and Perfadex groups (0.82). However, there were higher (p = 0.003) ATP in the SOE illuminated groups (4.61) compared with the non-illuminated groups (2.73). The PCr was also higher (p < 0.0001) in the SOE illuminated groups (1.89) compared with the non-illuminated groups (0.18). In conclusion, Perfadex in this experimental model was incapable of preserving the high energy phosphates in skeletal muscle during 5 hours of ischemia. Illumination with SOE at 634 nm improved the preservation potential, in terms of a positive effect on the energy status of the muscle cell.     

Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol

Commercial cellulase enzymes have been used in the food, detergent, and textile industries, and are potentially effective for processing biomass feedstocks. A survey was undertaken to identify major manufacturers/distributors of cellulases in the USA and to evaluate 13 representative commercial preparations for enzyme activity, protein concentration, and chemical composition. Samples were subjected to activity measurements using filter paper, carboxymethylcellulose, cellobiose, and p-nitrophenyl--d-glucopyranoside as substrates. To ascertain the microbial origin of the commercial preparations, Western blots utilizing monoclonal antibodies specific for Trichoderma reesei CBH I and Aspergillus niger -d-glucosidase were developed. Eleven of the cellulases tested were of T. reesei or T. viride origin and two were from A. niger.     

IL-1beta and LPS induce anorexia by distinct mechanisms differentially dependent on microsomal prostaglandin E synthase-1

Recent work demonstrated that the febrile response to peripheral immune stimulation with proinflammatory cytokine IL-1beta or bacterial wall lipopolysaccharide (LPS) is mediated by induced synthesis of prostaglandin E(2) by the terminal enzyme microsomal prostaglandin E synthase-1 (mPGES-1). The present study examined whether a similar mechanism might also mediate the anorexia induced by these inflammatory agents. Transgenic mice with a deletion of the Ptges gene, which encodes mPGES-1, and wild-type controls were injected intraperitoneally with IL-1beta, LPS, or saline. Mice were free fed, and food intake was continuously monitored with an automated system for 12 h. Body weight was recorded every 24 h for 4 days. The IL-1beta induced anorexia in wild-type but not knock-out mice, and so it was almost completely dependent on mPGES-1. In contrast, LPS induced anorexia of the same magnitude in both phenotypes, and hence it was independent of mPGES-1. However, when the mice were prestarved for 22 h, LPS induced anorexia and concomitant body weight loss in the knock-out animals that was attenuated compared with the wild-type controls. These data suggest that IL-1beta and LPS induce anorexia by distinct immune-to-brain signaling pathways and that the anorexia induced by LPS is mediated by a mechanism different from the fever induced by LPS. However, nutritional state and/or motivational factors also seem to influence the pathways for immune signaling to the brain. Furthermore, both IL-1beta and LPS caused reduced meal size but not meal frequency, suggesting that both agents exerted an anhedonic effect during these experimental conditions.     

Comparative study on enzymatic digestibility of switchgrass varieties and harvests processed by leading pretreatment technologies

Feedstock quality of switchgrass for biofuel production depends on many factors such as morphological types, geographic origins, maturity, environmental and cultivation parameters, and storage. We report variability in compositions and enzymatic digestion efficiencies for three cultivars of switchgrass (Alamo, Dacotah and Shawnee), grown and harvested at different locations and seasons. Saccharification yields of switchgrass processed by different pretreatment technologies (AFEX, dilute sulfuric acid, liquid hot water, lime, and soaking in aqueous ammonia) are compared in regards to switchgrass genotypes and harvest seasons. Despite its higher cellulose content per dry mass, Dacotah switchgrass harvested after wintering consistently gave a lower saccharification yield than the other two varieties harvested in the fall. The recalcitrance of upland cultivars and over-wintered switchgrass may require more severe pretreatment conditions. We discuss the key features of different pretreatment technologies and differences in switchgrass cultivars and harvest seasons on hydrolysis performance for the applied pretreatment methods.     

Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO₂, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.