Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160.

Hexose and pentose sugars from phosphoric acid pretreated sugarcane bagasse were co-fermented to ethanol in a single vessel (SScF), eliminating process steps for solid-liquid separation and sugar cleanup. An initial liquefaction step (L) with cellulase was included to improve mixing and saccharification (L+SScF), analogous to a corn ethanol process. Fermentation was enabled by the development of a hydrolysate-resistant mutant of Escherichia coli LY180, designated MM160. Strain MM160 was more resistant than the parent to inhibitors (furfural, 5-hydroxymethylfurfural, and acetate) formed during pretreatment. Bagasse slurries containing 10% and 14% dry weight (fiber plus solubles) were tested using pretreatment temperatures of 160-190°C (1% phosphoric acid, 10 min). Enzymatic saccharification and inhibitor production both increased with pretreatment temperature. The highest titer (30 g/L ethanol) and yield (0.21 g ethanol/g bagasse dry weight) were obtained after incubation for 122 h using 14% dry weight slurries of pretreated bagasse (180°C).

Optimizing the saccharification of sugar cane bagasse using dilute phosphoric acid followed by fungal cellulases.

A low level of phosphoric acid (1% w/w on dry bagasse basis, 160 degrees C and above, 10 min) was shown to effectively hydrolyze the hemicellulose in sugar cane bagasse into monomers with minimal side reactions and to serve as an effective pre-treatment for the enzymatic hydrolysis of cellulose. Up to 45% of the remaining water-insoluble solids (WIS) was digested to sugar monomers by a low concentration of Biocellulase W (0.5 filter paper unit/gWIS) supplemented with beta-glucosidase, although much higher levels of cellulase (100-fold) were required for complete hydrolysis. After neutralization and nutrient addition, phosphoric acid syrups of hemicellulose sugars were fermented by ethanologenic Escherichia coli LY160 without further purification. Fermentation of these syrups was preceded by a lag that increased with increased pre-treatment temperature. Further improvements in organisms and optimization of steam treatments may allow the co-fermentation of sugars derived from hemicellulose and cellulose, eliminating need for liquid-solid separation, sugar purification, and separate fermentations.

Expression of thrombomodulin and consequences of thrombomodulin deficiency during healing of cutaneous wounds.

Thrombomodulin is a cell surface anticoagulant that is expressed by endothelial cells and epidermal keratinocytes. Using immunohistochemistry, we examined thrombomodulin expression during healing of partial-thickness wounds in human skin and full-thickness wounds in mouse skin. We also examined thrombomodulin expression and wound healing in heterozygous thrombomodulin-deficient mice, compound heterozygous mice that have <1% of normal thrombomodulin anticoagulant activity, and chimeric mice derived from homozygous thrombomodulin-deficient embryonic stem cells. In both human and murine wounds, thrombomodulin was absent in keratinocytes at the leading edge of the neoepidermis, but it was expressed strongly by stratifying keratinocytes within the neoepidermis. No differences in rate or extent of reepithelialization were observed between wild-type and thrombomodulin-deficient mice. In chimeric mice, both thrombomodulin-positive and thrombomodulin-negative keratinocytes were detected within the neoepidermis. Compared with wild-type mice, heterozygous and compound heterozygous thrombomodulin-deficient mice exhibited foci of increased collagen deposition in the wound matrix. These findings demonstrate that expression of thrombomodulin in keratinocytes is regulated during cutaneous wound healing. Severe deficiency of thrombomodulin anticoagulant activity does not appear to alter reepithelialization but may influence collagen production by fibroblasts in the wound matrix.

Enzymatic cleavage of peptide-linked radiolabels from immunoconjugates.

We have incorporated peptides selected by combinatorial library [Peterson, J. J., and Meares, C. F. (1998) Bioconjugate Chem. 9, 618-626) into peptide-linked radiolabeled immunoconjugates of the form DOTA-peptide-antibody. Decapeptide linkers -GFQGVQFAGF- and -GFGSVQFAGF-, selected for cleavage by human liver cathepsin B, were rapidly digested in vitro when compared to the simple model tetrapeptide motif of the prototype -GGGF- [Li, M., and Meares, C. F. (1993) Bioconjugate Chem. 4, 275-283]. Cleavage properties of these library-selected substrates for cathepsin B compared favorably with decapeptide linkers -GLVGGAGAGF- and -GGFLGLGAGF-, which incorporate two of the most labile extended cathepsin B substrates from the literature. The decapeptide linker -GFGSTFFAGF-, selected from the library for cleavage by human liver cathepsin D, was rapidly digested by cathepsin D while the others were not.

Total solid-phase synthesis of 1,4,7,10-tetraazacyclododecane-N,N', N'',N'''-tetraacetic acid-functionalized peptides for radioimmunotherapy.

A convenient approach to the functionalization of peptides with the macrocyclic 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) moiety has been developed. Protected components (using tert-butyl or tert-butyloxycarbonyl groups) of both the peptide and the chelate were assembled on the same solid resin support. Deprotection and cleavage of the resin-bound DOTA-peptides were performed in one step using a trifluoroacetic acid cleavage mixture to yield free DOTA-peptide amides.

Subchondral metastasis: report of five cases.

Subchondral metastasis is a rare occurrence and poses a diagnostic dilemma as initial films may show a lytic lesion in the subchondral region often misinterpreted as being benign. We present five cases of subchondral metastasis as well as a review of the literature. In our cases, we present subchondral metastasis in the elbow, shoulder, and hip joints. All patients had pain over the affected joint and most presented with a lytic lesion in the subchondral bone. Three patients have died since presentation and two are doing well at last follow up visit. Subchondral metastasis is a rare entity, but it should be included in the differential of a lytic lesion in the subchondral bone.

Estimation of confidence intervals for area under the curve from destructively obtained pharmacokinetic data.

The area under the curve (AUC) of the concentration-time curve for a drug or metabolite, and the variation associated with the AUC, are primary results of most pharmacokinetic (PK) studies. In nonclinical PK studies, it is often the case that experimental units contribute data for only a single time point. In such cases, it is straightforward to apply noncompartmental methods to determine an estimate of the AUC. In this report, we investigate noncompartmental estimation of the AUC using the long-trapezoidal rule during the elimination phase of the concentration-time profile, and we account for the underlying distribution of data at each sampling time. For data that follow a normal distribution, the log-trapezoidal rule is applied to arithmetic means at each time point of the elimination phase of the concentration-time profile. For data that follow a lognormal distribution, as is common with PK data, the log-trapezoidal rule is applied to geometric means at each time point during elimination. Since the log-trapezoidal rule incorporates nonlinear combinations of mean concentrations at each sampling time, obtaining an estimate of the corresponding variation about the AUC is not straightforward. Estimation of this variance is further complicated by the occurrence of lognormal data. First-order approximations to the variance of AUC estimates are derived under the assumptions of normality, and lognormality, of concentrations at each sampling time. AUC estimates and variance approximations are utilized to form confidence intervals. Accuracies of confidence intervals are tested using simulation studies.

Comparison of 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-peptide-ChL6, a novel immunoconjugate with catabolizable linker, to 2-iminothiolane-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 in breast cancer xenografts.

Radioimmunotherapy using 131I-ChL6 antibody has shown promise in patients with breast cancer. To enhance this potential, a novel ChL6 immunoconjugate that is catabolizable and tightly binds 90Y and (111)In was developed. The immunoconjugate, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-peptide-ChL6, consists of the macrocyclic chelator DOTA linked to ChL6 by a peptide that is preferentially catabolized in the liver. The pharmacokinetic and dosimetric properties of the radioimmunoconjugates (RICs) (111)In- and 90Y-DOTA-peptide-ChL6 and (111)In- and 90Y-2-iminothiolane (2-IT)-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 were compared in athymic mice bearing HBT3477 human breast cancer xenografts. Each of the RICs was stable in vivo and concentrated well in the xenografts. Liver concentration, cumulative radioactivity (activity over time), and radiation dose of the DOTA-peptide-ChL6 RICs were one-third to one-half of those of the corresponding 2-IT-2-[p(bromoacetamido)benzyl]-DOTA-ChL6 RICs. Indium-111 RICs were imperfect tracers for corresponding 90Y RICs, although their pharmacokinetics and radiation dosimetries were similar. The results of this study were consistent with previously published in vitro data, which indicated that the peptide linker of DOTA-peptide-ChL6 was catabolized by cathepsin B. The cumulative activities and radiation doses to the liver of DOTA-peptide-ChL6 RICs were one-half of those of corresponding RICs with the 2-IT linker. Preliminary data from pilot studies in patients with breast cancer are in accord with these observations. These novel DOTA-peptide RICs seem to have excellent clinical potential for radioimmunotherapy associated with marrow transplantation, for which liver radiation is likely to be dose limiting for 90Y.

Cathepsin substrates as cleavable peptide linkers in bioconjugates, selected from a fluorescence quench combinatorial library.

Several extended peptide substrates for the human liver enzymes cathepsin B and cathepsin D have been selected as cleavable linkers for lysosomal proteolysis of bioconjugates. A one-bead-one-peptide combinatorial library of 9(4) fluorogenic substrates was employed. We designed this library to explore a set of substrates containing nonionizable/nonoxidizable groups to meet the requirements of prelabeling [Li et al. (1994) Bioconjugate Chem. 5, 101-104] as well as to yield stable conjugates whose preparation is straightforward.

Keratinocyte-specific expression of human thrombomodulin in transgenic mice: effects on epidermal differentiation and cutaneous wound healing.

BACKGROUND: Thrombomodulin is a cell-surface glycoprotein that regulates coagulation and fibrinolysis. Expression of thrombomodulin by epidermal keratinocytes is tightly regulated during squamous differentiation and cutaneous wound healing. METHODS: To determine the consequences of overexpression of thrombomodulin on squamous differentiation and wound healing in vivo, we expressed full-length human thrombomodulin in transgenic mice using the human keratin 14 promoter. Human thrombomodulin was detected in keratinocytes of transgenic mice by immunohistochemistry and protein C activation assays. Full-thickness cutaneous wounds were created on the dorsum of transgenic mice and nontransgenic littermates, and allowed to heal for up to 35 days. RESULTS: Transgenic mice had normal viability and appeared healthy up to one year of age. In the skin, human thrombomodulin was expressed in basal and suprabasal keratinocytes, with variable expression in the outer root sheath of hair follicles. Thrombomodulin activity in neonatal epidermis was 2.5- to 3-fold higher in transgenic mice than in nontransgenic littermates (p < 0.01). In cutaneous wounds, human thrombomodulin was expressed in migrating neoepidermal keratinocytes. No differences in keratinocyte migration or re-epithelialization were observed between transgenic and nontransgenic mice, but transgenic mice exhibited delayed collagen bundle deposition within the wound matrix. CONCLUSIONS: These findings demonstrate that keratinocyte thrombomodulin supports activation of protein C, and that thrombomodulin activity in epidermis can be increased by keratinocyte-specific expression of human thrombomodulin in transgenic mice. Expression of human thrombomodulin in keratinocytes does not impair normal squamous differentiation or re-epithelialization of cutaneous wounds, but may modulate collagen reconstitution of the wound matrix.

Extended storage of single-donor apheresis platelets in CLX blood bags: effect of storage on platelet morphology, viability and in vitro function.

A CLX (Cutter Laboratories, Berkeley, Calif.) bag system was evaluated for storage of single-donor apheresis platelets collected with the Haemonetics V-50 blood processor. Concentrates (n = 21) containing 3.9-5.2 x 10(11) platelets in 292 (+/- 41.8) ml were stored in two 1-liter bags for 7 days at 22 degrees C. pH was well maintained, declining from an initial pH of 7.0 (+/- 0.04) to 6.92 (+/- 0.20) after 7 days. Platelet morphology, response to a hypotonic stimulus and aggregation induced by paired agonists (epinephrine and ADP, or collagen) were also well-preserved. Concentrates with a wide variation of platelet yields (2.0 greater than or equal to 5.2 x 10(11), n = 43) also maintained pH (6.96 +/- 0.26), morphology and aggregation when stored for 7 days. All platelet concentrates (n = 64) were sterile at collection. Single-donor apheresis platelets may be stored in this bag system for up to 7 days.