Delignification of Wood Chips and Pulps by Using Natural and Synthetic Porphyrins: Models of Fungal Decay

Kraft pulps, prepared from softwoods, and small chips of birch wood were treated with heme and tert-butyl hydroperoxide in aqueous solutions at reflux temperature. Analyses of treated pulps showed decreases in kappa number (a measure of lignin content) from about 36 to less than 2, with concomitant increases in brightness (80% increase in the better samples). Analyses of treated wood chips revealed selective delignification and removal of hemicelluloses. After 48 h of treatment, lignin losses from the wood chips approached 40%, and xylose/mannose (hemicellulose) losses approached 70%, while glucose (cellulose) losses were less than 10%. Examination of delignified chips by transmission electron microscopy showed that the removal of lignin occurred in a manner virtually indistinguishable from that seen after decay by white rot fungi. Various metalloporphyrins, which act as biomimetic catalysts, were compared to horseradish peroxidase and fungal manganese peroxidase in their abilities to oxidize syringaldazine in an organic solvent, dioxane. The metalloporphyrins and peroxidases behaved similarly, and it appeared that the activities of the peroxidases resulted from the extraction of heme into the organic phase, rather than from the activities of the enzymes themselves. We concluded that heme-tert-butyl hydroperoxide systems in the absence of a protein carrier mimic the decay of lignified tissues by white rot fungi.     

In vitro toxicology of fumonisins and the mechanistic implications

The effects of fumonisins B₁FB₁, B₂(FB{2}), and the backbone of fumonisin B₁ remaining after hydrolysis of the tricarballylic groups with base (HFB₁) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK₁). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC₅₀ of FB₁=0.1 M), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC₅₀ for FB₁=35 M), and caused decreased cell proliferation as well. Higher doses (70 M) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation.     

Functional levels of mitochondrial anion transport proteins in non-insulin-dependent diabetes mellitus

The effect of non-insulin-dependent diabetes mellitus (i.e., NIDDM; type 2 diabetes) on the levels of functional mitochondrial anion transport proteins has been determined utilizing a chemically-induced neonatal model of NIDDM. We hypothesized that moderate insulin deficiency exacerbated by the insulin resistance, which is characteristic of NIDDM, would cause changes in mitochondrial anion transporter function that were similar to those we have previously shown to occur in insulin-dependent diabetes mellitus (i.e., IDDM; type 1 diabetes) (Arch. Biochem. Biophys. 280: 181–191, 1990). Our experimental approach consisted of the extraction of the pyruvate, dicarboxylate and citrate transport proteins from the mitochondrial inner membrane with Triton X-114 using rat liver mitoplasts (prepared from diabetic and control animals) as the starting material, followed by the functional reconstitution of each transporter in a proteoliposomal system. This strategy permitted the quantification of the functional levels of these three transporters in the absence of the complications that arise when such measurements are carried out with intact mitochondria (or mitoplasts). We found that experimental NIDDM did not cause significant changes in the extractable and reconstitutable specific (and total) transport activities of the pyruvate, dicarboxylate, and citrate transporters. These results are in marked contrast to our previous findings obtained using rats with IDDM and negated our hypothesis. The present results, in combination with our earlier findings, allow us to conclude that insulin plays an important role in the regulation of mitochondrial anion transporter function. Accordingly, in this model of NIDDM, where the level of insulin is not profoundly deficient, transporter function is unaltered, whereas in IDDM, where a profound insulinopenia exists, transporter function is altered. Furthermore, the present studies suggest that in the neonatal model of NIDDM the three mitochondrial transporters investigated are neither affected by, nor are they the sites of the well documented hepatic post-receptor insulin resistance which is characteristic of this disease.     

Lack of expression on cultured human T-lymphocytes of a T-cell antigen shared by the molt-4 cell line and normal human thymocytes

Summary Four hematopoietic cell lines (CCRF-CEM, HSB-2, MOLT-4, and RPMI-8402), derived from acute lymphoblastic leukemia and expressing T-cell surface markers (T-HCL), were studied with two specific anti-T-cell sera. The sera were raised in rabbits against human thymocytes (anti-HTY) and against T-cells cultured in the presence of conditioned medium derived from lymphocytes stimulated with PHA (anti-CTC). Both sera were absorbed to obtain a T-cell specific pattern of reaction and were further absorbed with normal peripheral blood lymphocytes or with each of the four T-HCL. The anti-HTY sera absorbed with CEM, 8402, and HSB-2 still reacted with MOLT-4. A similar pattern of reactivity was found only with the anti-CTC absorbed with 8402, whereas, after absorptions with the other cell lines, this antiserum was unreactive against MOLT-4. After absorption with normal peripheral blood lymphocytes, anti-HTY still reacted with thymocytes and MOLT-4 but was negative on CTC. In contrast, anti-CTC absorbed with peripheral blood lymphocytes (PBL) was negative on thymocytes and MOLT-4 but still reacted against CTC. Our data confirm the existence of a T-cell antigen (probably an early T-cell differentiation antigen) shared between thymus and MOLT-4. This antigen is not expressed on CTC, although these cells express an antigenic pattern more complex than PBL. Antisera to CTC represents a source of anti-T-cell sera free of contamination with antibodies to early thymus-related antigens but containing other T-cell-related specificities. Supported in part by Naval Medical Research and Development Command, Research Task No. ZF51.524.013.1025, and National Cancer Institute Contract No. Y01-CB-00319. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, National Research Council.     

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.     

Poly(methylene co-guanidine) coated alginate as an encapsulation matrix for urease

Urease was encapsulated within alginate beads, coated with poly(methylene co-guanidine) membranes via polyelectrolyte complexation. Membrane thickness increased with reaction time to 53 μm after 80 min, and to 59 μm with an increase in co-guanidine concentration from 2.5 to 20 mg ml⁻¹. A 70% mass and 31% activity yield of urease resulted following encapsulation. Although co-guanidine strongly inhibited freely soluble urease (I_0.5=5.8 μg ml⁻¹ co-guanidine), immobilization stabilized the enzyme against inactivation. Encapsulated activity declined as the polycation concentration used for membrane formation increased; however an activity loss of only 35% was observed when the co-guanidine concentration was as high as 5 mg ml⁻¹. Glucose protected against inactivation, with 0.5 increasing to 28.5 μg ml⁻¹ for the freely soluble enzyme. When the beads were coated with co-guanidine in the presence of glucose, encapsulated urease activity was fully retained.     

Diversification and biogeographic history of the Western Palearctic freshwater flatworm genus Schmidtea (Tricladida: Dugesiidae), with a redescription of Schmidtea nova

The freshwater flatworm genus Schmidtea is endemic in the Western Palearctic region, where it is represented by only four species, thus contrasting with the high species diversity of the closely related genus Dugesia within Europe. Although containing an important model species in developmental and regeneration research, viz. Schmidtea mediterranea, no evolutionary studies on the genus Schmidtea have been undertaken. For the first time, we present a well‐resolved molecular phylogenetic tree of the four species of the genus, inferred on the basis of two molecular markers, and provide also the first detailed morphological account of Schmidtea nova. The phylogenetic tree generated corroborates an earlier speciation hypothesis based on karyological data and points to chromosomal rearrangements as the main drivers of speciation in this genus. The high genetic divergence between the four species, in combination with previous dating studies and their current geographic distribution, suggests that Schmidtea could have originated in Laurasia but lost most of its diversity during the Oligocene. Thus, its present distribution pattern may be the result of the expansion of three of its four relictual species over Europe, probably after the Pleistocene glaciations. Our detailed morphological study of S. nova revealed that it shows a number of remarkable features: interconnected testis follicles, parovaria, an ejaculatory duct exiting into the primary as well as the secondary seminal vesicle by means of a nipple, and the wall of the distal section of the ejaculatory duct being sclerotic or chitinized.     

Reliability and accuracy of handheld stopwatches compared with electronic timing in measuring sprint performance

This study assessed reliability of split times obtained by handheld stopwatches (HHSs) compared with electronic timing (ET) during a 200-m sprint. Two HHS timing methods were compared with ET: single-split timers (SST) and multiple-split timers (MST). Twenty-six timers without previous experience were given instruction and completed practice trials until good agreement was achieved between ET and HHS. Trained runners (8 males, 10 females) were timed for each 25-m interval on a standard 200-m course. Repeated-measures analysis of variance and intraclass correlation models were used to determine reliability. A total of 248 split times were analyzed. No significant differences were found between the three timing methods (p > 0.99), and calculated intraclass correlation values were high (0.988). Mean error between SST, MST, and ET (-0.04 +/- 0.24 and -0.05 +/- 0.24 seconds, respectively) indicated faster HHS times, though not significantly. However, absolute errors were considerably larger (0.15 +/- 0.20 and 0.16 +/- 0.19 between SST, MST, and ET, respectively). The HHS-recorded splits were faster than ET in 67.3% of splits and slower in 29.4%. The distribution of errors made the development of a reliable correction factor to convert HHS to ET impossible. It was concluded that on the basis of the small mean error and high intraclass correlations, the use of HHSs may be a viable alternative to ET in collecting group data. However, on the basis of the absolute error between HHS and ET, when high degrees of precision are required, ET should be used, and reliable correction of HHS to ET values is not possible. It was further concluded that HHS times should be reported without attempting correction and interpreted in light of the shortcomings of the HHS method.