Probiotics in Goat Milk Products: Delivery Capacity and Ability to Improve Sensory Attributes.

Dairy foods, particularly those of bovine origin, are the predominant vehicles for delivery of probiotic bacteria. Caprine (goat) milk also possesses potential for successful delivery of probiotics, and despite its less appealing flavor in some products, the use of goat milk as a probiotic carrier has rapidly increased over the last decade. This review reports on the diversity, applicability, and potential of using probiotics to enhance the sensory properties of goat milk and goat milk-based products. A brief conceptual introduction to probiotic microorganisms is followed by an account of the unique physicochemical, nutritive, and beneficial aspects of goat milk, emphasizing its advantages as a probiotic carrier. The sensory properties of probiotic-enriched goat milk products are also discussed. The maintenance of probiotic viability and desirable physicochemical characteristics in goat milk products over shelf life is possible. However, the unpleasant sensory features of some goat milk products remain a major disadvantage that hinder its wider utilization. Nevertheless, certain measures such as fortification with selected probiotic strains, inclusion of fruit pulps and popular flavor compounds, and production of commonly consumed tailor-made goat milk-based products have potential to overcome this limitation. In particular, certain probiotic bacteria release volatile compounds as a result of their metabolism, which are known to play a major role in the aroma profile and sensory aspects of the final products.

Ex vivo/in vitro absorption of STW 5 (Iberogast) and its extract components.

To correlate the pharmacological effects of the fixed herbal combination STW 5 (Iberogast) containing nine extract components with its confirmed clinical efficacy, ex vivo/in vitro absorption tests were performed. For the investigation, the everted gut sac technique and, in a pilot study, the Caco-2-cell model were used. The absorption rate of the extracts was determined by measuring characteristic marker substances of each of the individual extracts using HPLC or GC techniques. The results allow us to conclude that the investigated substances from STW 5 possess a good bioavailability, which is in accordance with the rapid onset of the therapeutic efficacy and explains its known pharmacological effects and clinical efficacy in terms of multiple drug action and multi-target therapy, respectively.

Colgajo miocutáneo de glúteo mayor en isla con cierre V en Y para la cobertura de úlceras isquiáticas / Island miocutaneous gluteus maximus flap for coverage of ischiatic ulcers

Las úlceras en la región isquiáticas ocurren por la permanencia en posición de sentado durante períodos prolongados. Son las úlceras con la mayor recidiva y presencia de lechos ulcerosos múltiples y sinuosos. Esto ocasiona hospitalización prolongada, aseos quirúrgicos repetidos, antibioterapia múltiple y retraso de la rehabilitación. La cobertura se puede realizar con colgajos miocutáneos de glúteo mayor, bíceps femoris, gracilis, tensor de fascia lata, semimembranoso y semitendinoso, entre otros. El colgajo miocutáneo de glúteo mayor realizado para la cobertura de úlceras isquiáticas, es actualizado en este trabajo y se incorpora como variante la utilización de una isla cutánea irrigada por perforantes, que avanza al lecho con cierre V en Y sin tensión. Nuestro objetivo es demostrar que el colgajo de glúteo mayor sería de primera elección en la cobertura de úlceras isquiáticas. Proponemos como variante quirúrgica la liberación completa del músculo, proveyendo un amplio eje de rotación, y una isla de piel sobre él, produciendo un cierre sin tensión. Todo esto reduciría la morbilidad y recidiva. Se realizaron 13 colgajos miocutáneos de glúteo mayor de avance y rotación con cierre V en Y para la cobertura de úlceras isquiáticas de presión grado III y IV. Las patologías asociadas fueron tetraplejia y paraplejia por lesión medular y paraplejia espástica familiar. El seguimiento fue de 6 meses hasta 7 años. Hasta la fecha no se ha documentado morbilidad ni recidiva. En nuestra experiencia el colgajo miocutáneo de glúteo mayor seria de elección para cierre de úlceras isquiáticas por su volumen de relleno e importante irrigación. El cierre sin tensión estaría dado por la liberación completa del músculo produciendo un amplio eje de rotación, y por una isla de piel que se transporta sobre él (AU)

The ischiatic ulcers develop as a consecuense of long sitting position. This kind of ulcers relapse in a great percentage and are a challenge for clinicians. Most of the time causes long hospital stays, multiple surgical procedures and complex antimicrobial therapy. Coverage can be done with several myocutaneous flaps, including gluteus maximus, biceps femoris, gracilis, tensor fascia lata, semitendinosus (AU)

Computer assisted retraining of attentional impairments in patients with multiple sclerosis.

OBJECTIVE: To evaluate the efficacy of a computer based retraining of specific impairments of four different attentional domains in patients with multiple sclerosis. METHODS: Twenty two outpatients with multiple sclerosis received consecutively a specific training comprising 12 sessions in each of the two most impaired attention functions. The baseline of attentional deficits, the performance after each training period, and the course of performance in the next nine weeks was assessed by a computerised attention test battery. Additionally, the impact of the training on daily functioning was evaluated with a self rating inventory. RESULTS: Subgroups of patients with multiple sclerosis showing different patterns of attentional impairment could be separated. Significant improvements of performance could almost exclusively be achieved by the specific training programmes. The increase of performance remained stable for at least nine weeks. For quality of life patients reported less attention related problems in everyday situations. CONCLUSIONS: In patients with multiple sclerosis it seems worthwhile to assess attentional functions in detail and to train specific attention impairments selectively.

The herpesvirus protease: mechanistic studies and discovery of inhibitors of the human cytomegalovirus protease.

The herpesvirus protease is a recently identified enzyme which is essential for viral replication. It is found in all herpesviruses and offers a new molecular target for therapeutic intervention. Its genomic structure has recently been described and consists of a large open reading frame which encodes a fusion protein containing an amino-terminal protease domain in-frame with a carboxyl-terminal "assembly protein-like" domain. Auto-processing releases the amino-terminal protease as a maturational enzyme. The herpesvirus protease has been characterized as a novel serine protease. Four surface accessible sulfhydryl groups have been identified in the human cytomegalovirus (HCMV) protease. Utilizing a fluorogenic DABCYL-EDANS substrate assay, directed screening has identified a class of sulfhydryl-modifying benzimidazolylmethyl sulfoxides which inhibits recombinant HCMV protease. Site-directed mutagenesis studies suggest oxidative modification of surface-accessible HCMV protease Cys138 (and possibly Cys161) by this class of inhibitors. The benzimidazolylmethyl sulfoxide 1 inhibits HCMV protease (IC50 = 1.9 microM), exhibits selectivity vs. mammalian serine proteases, and exhibits antiviral activity in an HCMV infected cell culture assay.

Three-dimensional structure of human cytomegalovirus protease.

Herpesviruses encode a serine protease that specifically cleaves assembly protein. This protease is critical for replication, and represents a new target for antiviral drug design. Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 angstroms resolution. The structure reveals a unique fold and new catalytic strategy for cleavage. The monomer fold of the enzyme, a seven-stranded beta-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin. The serine nucleophile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157. Dimerization, which seems to be necessary for activity, is observed in the crystals. Correlations of the structure with the sequences of herpesvirus proteases suggest that dimerization may confer specificity and recognition in substrate binding.

Intraocular production of a cytokine (CINC) responsible for neutrophil infiltration in endotoxin induced uveitis.

AIMS/BACKGROUND: The subcutaneous injection of bacterial endotoxin in Lewis rats produces an acute intraocular inflammation evolving over a 24 hour period. This endotoxin induced uveitis (EIU) is characterised by a biphasic protein exudation and a cellular infiltrate composed of macrophages and polymorphonuclear neutrophils (PMNs). This model was used to study the mechanism of cellular infiltration in ocular inflammation. METHODS: EIU was induced by a subcutaneous injection of lipopolysaccharide (LPS) (S typhimurium) at 350 micrograms/kg. The levels of cytokine induced neutrophil chemoattractant (CINC) were measured every 2 hours in the serum and in the aqueous humour by ELISA. The intraocular inflammation was quantified by protein measurement and leucocyte counting. RESULTS: The kinetics of CINC production in the systemic circulation showed a rapid rise, peaking 2 hours after LPS injection, followed by a progressive decline over the next 8 hours. In the eye, the CINC levels increased above the serum levels 10 hours after EIU induction corresponding to the time of cellular infiltration. When leucocyte entry in the eye was inhibited by 56% and 64% with an antiadhesion molecule antibody, there was only a slight reduction in the aqueous humour CINC levels of 9% and 16%, respectively, indicating that CINC was produced by ocular tissue cells. The specific effect of CINC in the eye was confirmed when a direct intraocular injection of 250 ng of purified CINC was followed by significant PMN infiltration, in the absence of protein exudation. CONCLUSION: The data indicate that the production of the CINC chemotactic factor by ocular tissue participates in the inflammatory reaction in EIU.

Intratracheal injection of endotoxin and cytokines. IX. Contribution of CD11a/ICAM-1 to neutrophil emigration.

Intratracheal injection of endotoxin [lipopolysaccharide (LPS)] in rats causes acute inflammation characterized by the emigration of neutrophils (PMNs) into the bronchoalveolar airspace. Antibody to PMN adhesion molecule CD11a inhibited LPS-initiated PMN accumulation in bronchoalveolar lavage (BAL) fluid by 32% (P < 0.001). Antibody to the endothelial CD11a counterreceptor intercellular adhesion molecule-1 (ICAM-1) inhibited LPS-initiated PMN accumulation in BAL fluid by 66% (P < 0.0001). Combined antibody blockade of ICAM-1 and the C-X-C chemokine cytokine-induced neutrophil chemoattractant (CINC) inhibited LPS-initiated PMN emigration by 80%, significantly more than antibody against either ICAM-1 or CINC alone. To study the relative contribution of alveolar macrophages and PMNs to intra-alveolar tumor necrosis factor (TNF), the LPS-induced TNF in BAL fluid was measured after depletion of circulating PMNs with a cytolytic antibody to CD18. Although the anti-CD18 antibody completely abrogated LPS-initiated PMN emigration into BAL fluid, TNF levels in BAL fluid were unaffected, suggesting that alveolar macrophages are the predominant cellular source of LPS-induced TNF production. In conclusion, 1) CD11a, ICAM-1, and CINC play major roles in the LPS-initiated emigration of PMNs into the bronchoalveolar space, and 2) the TNF that drives ICAM-1 and CINC expression is derived largely from alveolar macrophages rather than PMNs.

Chemokine gene expression in anti-glomerular basement membrane antibody glomerulonephritis.

Chemokines may be important in the pathogenesis of glomerular leukocyte infiltration in antiglomerular basement membrane (GBM) antibody (Ab) glomerulonephritis (GN). We studied the expression of the C-C chemokines [macrophage inflammatory protein (MIP)-1 alpha, monocyte chemotactic protein (MCP)-1, and RANTES] and C-X-C chemokines [platelet factor 4 (PF4), interferon-inducible protein of 10 kDa (IP-10), MIP-2, and cytokine-induced neutrophil chemoattractant (CINC)] at 30 min, 3, 6, 9, 15, and 24 h after induction of heterologous-phase anti-GBM Ab GN in Lewis rats. There was a rapid induction of CINC, MIP-2, MCP-1, and MIP-1 alpha mRNAs in the glomeruli of nephritic rats 30 min after administration of the anti-GBM Ab, whereas increases in PF4 and IP-10 mRNAs were not seen until 3 h. The mRNA expression of PF4, MIP-1 alpha, MIP-2, and IP-10 peaked at 3 h, whereas CINC and MCP-1 peaked at 6 and 15 h, respectively. By comparison, the expression of RANTES mRNA in rats with anti-GBM Ab GN did not differ from those of control rats. These changes in chemokine gene expression were associated with glomerular polymorphonuclear leukocytes (PMN) and monocyte/macrophage infiltration which peaked at 3 h (20.8 +/- 11.1 PMN/glomerulus) and 24 h (8.2 +/- 1.0 ED-1 cells/glomerulus), respectively. The administration of dexamethasone suppressed glomerular chemokine mRNA expression (60-98%) at both 3 and 15 h, which was associated with a 50-100% reduction in glomerular PMN and monocyte/macrophage infiltration, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Intratracheal administration of endotoxin and cytokines. VI. Antiserum to CINC inhibits acute inflammation.

Cytokine-induced neutrophil chemoattractant (CINC), a chemotactic molecule of the interleukin (IL)-8 family, is known to be induced in the rat in response to tumor necrosis factor (TNF), IL-1, and lipopolysaccharide (LPS). Intratracheal injection of endotoxin (LPS) is shown to cause CINC mRNA expression in pulmonary tissue, peaking after 2 h, and CINC protein expression in bronchoalveolar lavage (BAL) fluid, peaking after 2-4 h. Intratracheal injection of synthetic CINC causes acute inflammation that is abrogated by coinjection of antiserum to purified natural rat CINC. Intratracheal injection of antiserum to CINC inhibits intratracheal LPS- and IL-1-induced neutrophil emigration into BAL fluid by approximately 60-70%. Despite the anti-inflammatory activity of anti-CINC antiserum, TNF is elevated in the lavage fluid of rats receiving anti-CINC, suggesting that CINC may act in a negative feedback loop to downregulate TNF expression. Intratracheal injection of antiserum to CINC combined with intravenous injection of anti-E-selectin antibody inhibits intratracheal LPS- and IL-1-induced neutrophil emigration into BAL fluid by approximately 75-85%. CINC-mediated chemotactic activity and E-selectin-mediated adherence of neutrophils to endothelium contribute significantly to the pathogenesis of LPS-initiated acute inflammation.

Activity of two-chain recombinant human cytomegalovirus protease.

The human cytomegalovirus UL80 protease was expressed in Escherichia coli and purified by metal-chelate chromatography using a histidine tag engineered at the amino terminus. Cleavage of the 30-kDa protease at an internal site, VEA/A144, resulted in the recovery of 16- plus 14-kDa two-chain protease. The amino-terminal 16-kDa chain and the carboxyl-terminal 14-kDa chain remained associated as an active enzyme that was modified specifically at Ser132 on the 16-kDa chain by [3H]diisopropyl fluorophosphate. Disruption of the cleavage site by mutation from VEA/A to AEA/A facilitated the recovery of active 30-kDa one-chain enzyme that could be similarly modified at Ser132 by [3H]diisopropyl fluorophosphate. Both one- and two-chain enzymes cleaved recombinant assembly protein at the maturation site, VNA/S, and a peptide, GVVNASARL, mimicking this site. Internal processing does not inactivate the protease but forms a two-chain enzyme that retains activity.

Cytokine-induced neutrophil chemoattractant mediates neutrophil influx in immune complex glomerulonephritis in rat.

Chemokines are a family of cytokines whose participation in inflammation in vivo remains to be established. Using the rat model of anti-glomerular basement membrane (GBM) nephritis, we found that mRNA for the chemokine CINC (cytokine-induced neutrophil chemoattractant) was induced in the kidney, and the corresponding protein was elaborated by isolated inflamed glomeruli. Production of CINC by glomeruli was unaffected by complement- or leukocyte-depletion prior to disease induction. Cytokines which induce CINC expression in renal cells (TNF-alpha and IL-1 beta) were also expressed in the kidney during glomerular inflammation. TNF-alpha production, unlike CINC, was complement and leukocyte dependent. In vivo administration of anti-CINC, but not anti-human IL-8, IgG selectively attenuated the influx of PMNs into the glomerulus and commensurately diminished proteinuria. The participation of CINC was not tissue-specific: anti-CINC IgG also diminished the influx of PMNs in dermal immune complex inflammation. In sum, we propose that glomerular immune complex deposition/complement activation leads to cytokine production which results in CINC expression by endogenous glomerular cells. The CINC produced plays a contributory role in the influx of PMNs into the glomerulus in the context of the activation of other inflammatory mediators. These results suggest a potential role for CINC homologues, IL-8 and the GRO family of chemokines, in human immune complex-mediated disease.

The influence of platinum drugs on the radiation response of rat kidneys.

The influence of a single bolus injection of platinum drugs on the radiation sensitivity of the kidneys was investigated in WAG/Rij rats. Drugs employed were cis-diammine-dichloroplatinum(II) (cisplatin, CDDP), cis-diammine-1,1-cyclobutanedicarboxylate platinum(II) (carboplatin, CBDCA) and cis-dichloro,trans- dihydroxy-bis-isopropylamine platinum(IV) (iproplatin, CHIP). Both kidneys were irradiated with a range of single X-ray doses while drugs were administered at 1 day or 1 week before irradiation. Maximum tolerated drug doses (defined as the LD1, the dose resulting in a mortality of 1%) were given. Damage inflicted upon the kidneys was monitored by determination of several parameters indicative of kidney function. Isoeffective radiation doses were calculated from these data for each treatment group at 4-8-week intervals up to 80 weeks following treatment. At each assay time, dose modifying factors (DMF) were calculated for each drug/radiation combination. The mean DMFs were highest for CDDP: approximately 1.6. Those for CBDCA and CHIP were lower: approximately 1.1 and 1.2, respectively. The CHIP DMFs were significantly different from unity. When the radiation was given in 4 or 8 daily fractions (4 fractions/week) the DMFs for CDDP were identical to those obtained with single doses. For CBDCA and CHIP, however, the DMFs after fractionated treatments were not significantly different from unity. Analysis in terms of the linear-quadratic (LQ) model indicated that not one of the three drugs had an effect on the alpha/beta ratio, and hence on the fractionation sensitivity of the rat kidney. Consequently, if these data are extrapolated to the clinical setting, the administration of these drugs at the maximum tolerated dose preceding a fractionated radiation treatment should not be expected to result in extra, unexpected, radiation toxicity of the kidney.

Investigation of possible autocrine functions for rat GRO/CINC (cytokine-induced neutrophil chemoattractant).

Rat cytokine-induced neutrophil chemoattractant (CINC) is an eight kilodalton polypeptide originally purified from media conditioned by interleukin-1 beta stimulated 52E, an epithelioid clone derived from normal rat kidney (NRK) cells. Using a fibroblastic clone of the NRK cells, 49F, we found expression of the CINC gene to be induced by either serum or cytokines in growth-arrested cultures within 1 hour of stimulation. There was no observable CINC expression in exponentially growing cells in the absence of cytokine stimulation. CINC protein had no significant effect on 3H-thymidine incorporation or growth rate of NRK49F. We have observed that CINC is constitutively produced by some transformed NRK cells, clone RC20, suggesting an association with the expression of a transformed phenotype. Unlike the parent 49F, RC20 cells are capable of growth in soft agar and serum-free media and form highly metastatic tumors in nude mice. We have examined the possible autocrine functions of CINC and its possible links to the expression of the transformed phenotype by these cells. The use of a blocking CINC polyclonal antibody demonstrated that CINC did not function as an autocrine growth factor for RC20. Though CINC is a potent chemoattractant for neutrophils, it did not induce migration of either RC20 or 49F cells. CINC only moderately promoted adhesion of RC20 cells when used as a matrix protein. These data do not support the hypothesis that production of CINC by the RC20 cells provides an obvious advantage for the transformed cells constitutively producing it.

High-level expression of cytokine-induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: purification and production of blocking antibodies.

Significant levels of cytokine-induced neutrophil chemoattractant (CINC) were found in serum-free medium conditioned by a highly metastatic rat cell line, RC20. To study CINC's role in inflammation and metastasis, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits. CINC was a potent chemoattractant for rat neutrophils (EC-50 0.5 nM). A fusion protein of glutathione-S-transferase and CINC (GST-CINC) was produced in E. coli. Anti-CINC polyclonal IgG was purified from immune goat and rabbit sera by protein A and GST-CINC affinity chromatography. Both goat and rabbit anti-CINC antibody preparations at 4 micrograms/mL (an 11-fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat neutrophil chemotaxis assay. These antibodies have been used to develop a sensitive immunoassay for CINC. The availability of large amounts of affinity-purified blocking anti-CINC antibody will allow investigations into the role played by CINC in rodent inflammation models and in the metastasis of RC20 cells.

Cross-competition for binding of alpha 1-antitrypsin (alpha 1 AT)-elastase complexes to the serpin-enzyme complex receptor by other serpin-enzyme complexes and by proteolytically modified alpha 1 AT.

The serpin-enzyme complex (SEC) receptor recognizes a pentapeptide neo-domain of alpha 1-antitrypsin (alpha 1 AT)-elastase complexes and, in so doing, mediates internalization and intracellular catabolism of the macromolecular complex, mediates an increase in synthesis of alpha 1 AT, and elicits neutrophil chemotactic activity. In previous studies we have shown that this pentapeptide domain is highly conserved among members of the serpin family and that binding of a synthetic peptide corresponding to this region (125I-peptide 105Y, SIP-PEVKFNKPFVYLI, based on alpha 1 AT sequence 359-374) to HepG2 cells is blocked by several serpin-enzyme complexes. To determine whether the SEC receptor is the primary HepG2 cell surface binding site for these serpin-enzyme complexes, we examined the capacity for serpin-enzyme complexes to compete with each other for binding to the SEC receptor. The results indicate that binding of 125I-elastase-alpha 1 AT complexes is blocked by thrombin-antithrombin III (ATIII), thrombin-heparin cofactor II, and cathepsin G-alpha 1-antichymotrypsin (alpha 1 ACT) complexes. Moreover, unlabeled elastase-alpha 1 AT complexes compete for binding of 125I-thrombin-ATIII, 125I-thrombin-heparin cofactor II, and 125I-cathepsin G-alpha 1 ACT complexes. Preformed soluble tissue plasminogen activator-plasminogen activator inhibitor 1 complexes also compete for binding of elastase-alpha 1 AT complexes to the SEC receptor but do so to a less effective extent, probably because of a less favorable pentapeptide sequence for binding to the SEC receptor. Under conditions in which these serpin-enzyme complexes would be expected to bind to the SEC receptor there is an increase in synthesis of alpha 1 AT but not in synthesis of ATIII or alpha 1 ACT. Proteolytically modified alpha 1 AT also competes for binding of 125I-elastase-alpha 1 AT complexes to the SEC receptor and vice versa. The purified 51-kDa amino-terminal fragment of alpha 1 AT does not compete for binding of 125I-elastase-alpha 1 AT complexes, indicating that the pentapeptide neodomain in the 4-kDa carboxyl-terminal fragment is sufficient for binding to the SEC receptor.

Oligosaccharides at each glycosylation site make structure-dependent contributions to biological properties of human tissue plasminogen activator.

The effect of altering oligosaccharide structures at sites 184 and 448 of tissue plasminogen activator (tPA) has been examined. Alteration to high-mannose forms at sites 184 and 448 was accomplished by the growth of cells in the presence of deoxymannojirimycin (dMM). Modification to neutral, unsialylated forms at these sites was achieved by neuraminidase treatment of control preparations of tPA. Oligosaccharides at site 117 were not markedly affected by either treatment because structures at this site are high-mannose and not sialylated in untreated preparations. The effect on enzymatic activity and on a related property, lysine affinity, was determined. dMM treatment was found to increase both the lysine affinity and catalytic activity of tPA. Neuraminidase treatment increased enzyme activity, but was without effect on affinity for lysine. To evaluate the effects of alterations at site 184 and site 448, the catalytic activity and lysine affinity of type I and type II tPA were monitored individually. In the dMM-treated sample, type I tPA (with sugars at sites 117, 184 and 448) was found to have 2- to 3-fold increased catalytic activity and an affinity for lysine which was greater than that of type I from untreated preparations, but less than that of control type II tPA (containing sugar only at sites 117 and 448). In neuraminidase-treated type I, catalytic activity was also enhanced but lysine affinity remained unchanged. Type II from dMM- and neuraminidase-treated preparations had catalytic activity that was increased approximately 1.5-fold compared to untreated controls, whereas affinity for lysine was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Time- and sequence-dependent responses to cisplatin and radiation in the rat kidney.

The influence of time interval and sequence between administration of cisplatin and a radiation dose was studied in the rat kidney. A dose of 10.5 Gy X-rays was given to both kidneys, preceded or followed by a single dose of cisplatin. Two separate experiments were performed. In the first experiment 6.0 mg/kg cisplatin was given, in the second experiment the drug dose was 5.5 mg/kg. A range of time intervals was introduced between administration of drug and radiation, from 7 to 1 days, 12 to 1 h, and 30 to 0 min. Control animals received either modality alone, or were left untreated. Cisplatin alone caused tubular function to decrease very quickly and to remain permanently altered. Changes in glomerular function were only detected after 30 weeks following the higher drug dose. X-rays alone caused measurable alterations in both glomerular and tubular function after 16 weeks. In the combined treatment the influence of time and sequence was significant. If cisplatin was given at 7 to 1 days before X-rays the effect of time was minimal. Administration of cisplatin 12 h to 15 min before irradiation resulted in an increase of radiation damage with decreasing time interval. Total damage sharply decreased when both modalities were given at the same time, and decreased further with increasing time between irradiation and drug administration. It is suggested that in the tubular cells free cisplatin or one of its hydrolysis products may interact with radiation-induced damage, e.g. by interference with repair of sublethal or potentially lethal damage.

Effect of peptides on the inactivation of tissue plasminogen activator by plasminogen activator inhibitor-1 and on the binding of tissue plasminogen activator to endothelial cells.

The effectiveness of tissue plasminogen activator (tPA) in thrombolytic therapy is dependent upon the rate at which therapeutically administered tPA reaches the clot site and the proportion of that tPA which is enzymatically active. Interactions between tPA and its main plasma inhibitor (PAI-1) and between tPA and the endothelial cells lining blood vessels are two factors which may limit efficacy. In an attempt to identify the regions of the tPA molecule involved in these interactions, we have examined a series of synthetic peptides with amino acid sequences corresponding to different regions of the tPA molecule for their ability to protect tPA from inactivation by PAI-1 and for their ability to reduce the binding of tPA to endothelial cells. Three peptides were identified which were especially effective at maintaining tPA activity in the presence of PAI-1 and three others were found which had a lesser effect. These same peptides were also found to inhibit the binding of tPA to endothelial cells. This suggests that the same regions of the tPA molecule are involved in both processes. None of the peptides inhibited the binding of tPA to fibrin. These peptides may serve as models for the development of agents for enhancing the activity of both endogenous tPA and of tPA administered in thrombolytic therapy.

Secretion of active kringle-2-serine protease in Escherichia coli.

Active human tissue plasminogen activator variant kringle-2-serine protease (K2 + SP domains; referred to as MB1004) was synthesized as a secreted protein in Escherichia coli, isolated, and characterized. MB1004 is a relatively large and complex protein, approximately 38 kDa in size and containing nine disulfide bonds. MB1004 without a pro region was secreted into the periplasm of E. coli by fusing the protein to the PhoA leader peptide expressed from the tac promoter. Approximately 1% (20 micrograms/L broth) of the secreted MB1004 was purified from E. coli homogenates as a soluble, active enzyme by using a combination of lysine and Erythrina inhibitor affinity chromatography. Purified MB1004 was monomeric and single-chain, and the N-terminus was identical with the predicted amino acid sequence. The specific activity of purified MB1004 from E. coli was compared against the equivalent recombinant material purified from mammalian cells that was naturally glycosylated (MB1004G) or deglycosylated after treatment with N-glycanase (MB1004N). Results from four different in vitro assays showed that MB1004 and MB1004N had similar activities. Both exhibited 4-12-fold higher specific activity than MB1004G in plasminogen activation assays. These results suggest that an inaccurate picture of specific activity can be obtained if the effects of glycosylation are not considered. By utilization of secretion in E. coli, nonglycosylated MB1004 was purified without in vitro refolding and was shown to be suitable for structure-function studies.