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Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy. Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the promoter choice.

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Diaryl-substituted bicyclic amines are a scarcely investigated class of compounds. Only few of them are described and their biological activities are reported poorly. During our work in the field of heterocyclic chemistry, we found that 4-dialkylaminobicyclo[2.2.2]octan-2-ones and -ols show antiprotozoal properties against Plasmodium falciparum K(1) and Trypanosoma brucei rhodesiense, the causative organisms of Malaria tropica and of Human African Trypanosomiasis. Therefore, we synthesized over 200 derivatives in order to investigate their antitrypanosomal and antiplasmodial activities as well as their cyctotoxicity using in vitro microplate assays. Even if the target and the mechanism of action of these compounds are still unknown, we can at least provide several structure-activity relationships for this interesting class of compounds. Moreover, we achieved a distinct improvement of their antiplasmodial and antitrypanosomal properties.

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BACKGROUND: Delayed graft function (DGF) is a problem in kidney transplantation and cold ischemia has been identified as a risk factor. Perfluorocarbons (PFC) have an enhanced ability to dissolve and release oxygen. We evaluated histologically and a number of molecular changes induced by ischemia in stored kidneys with University of Wisconsin (UW) and PFC-based preservation solutions (PFC-UW). MATERIALS AND METHODS: ACI rats were used as kidney donors. UW (control group) or PFC-UW (study group) preservation solutions were used for kidney perfusion. All kidneys were stored at 4 degrees C for 12, 24, and 36 hours. After this time, intragraft histologic evaluation as well as mRNA HO-1 and iNOS levels were also analyzed. RESULTS: In the kidneys stored at 24 hours, mRNA HO-1 levels were elevated in the study group when compared with the control and mRNA iNOS was decreased. CONCLUSION: We observed overexpression of HO-1 and underexpression of iNOS in the kidney tissue stored with PFC-UW solution at 24 hours. These preliminary data suggest that increasing oxygen delivery by PFC added to the perfusion solution triggers cytoprotective mechanism in kidney transplantation.

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New 4-amino-6,7-diphenylbicyclo[2.2.2]octane derivatives, esters of bicyclo[2.2.2]octan-2-ols and O-methyl oximes of bicyclo[2.2.2]octan-2-ones were synthesised. Their activities against Trypanosoma brucei rhodesiense (STIB 900) and their activity against the K1 strain of Plasmodium falciparum (resistant to chloroquine and pyrimethamine) were determined by use of microplate assays. The cytotoxicity was assessed using L6 cells. The antiprotozoal activities of the new compounds are compared with those of former prepared derivatives and drugs in use. Structure-activity relationships are discussed.

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BACKGROUND: Autoimmune diabetes can be prevented in animal models by hypoallergenic diets. Moreover, in animal models, oral administration of insulin can suppress the development of autoimmune diabetes and clinical trials on prevention of human Type 1 diabetes by oral administration of insulin are already taking place. However, it has been reported that autoimmune diabetes can be induced by oral administration of an auto-antigen (insulin), and great caution is therefore warranted when applying the oral tolerance approach to prevent Type 1 diabetes. AIM: To evaluate the effect of orally administered insulin on the development of autoimmune diabetes in non-obese diabetic mice fed a hypoallergenic diet. METHODS: Four groups of mice were fed regular mouse chow (group 1, control mice), hypoallergenic diet, using the hydrolyzed infant formula Pregistimil (group 2), and Pregistimil with oral insulin (4 U/l of drinking water, group 3; and 8 U/l of drinking water, group 4). RESULTS: At 210 days of age, 11/20 (55%) mice in group 1 developed diabetes. In contrast, none of the mice from the Pregistimil-fed groups (0/16, 0/14, 0/17) developed the disease (p<0.001). The incidence of infiltrated islets and the severity of insulitis, at age 90 days, was significantly lower in mice fed with the hypoallegenic diet than in controls (p=0.028). CONCLUSIONS: In NOD mice fed a diet that prevents the development of diabetes, oral insulin supplementation appears to be safe, since it does not promote the development of clinical diabetes.

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Two experiments were conducted to determine independent effects of BW and DE intake on body composition and the partitioning of retained body energy between lipid and protein in pigs with high lean tissue growth potentials and when energy intake limited whole-body protein deposition. In a preliminary N-balance experiment involving 20 entire male pigs at either 30 or 100 kg BW, it was established that whole-body protein deposition increased linearly (P < 0.05) with DE intake at both BW. These results indicate that DE intake controlled whole-body protein deposition and that these pigs did not achieve their maximum whole-body protein deposition when fed semi-ad libitum. In the main serial slaughter experiment, 56 pigs, with a BW of 15 kg, were assigned to one of four DE intake schemes and slaughtered at 40, 65, 90, or 115 kg BW. Within DE intake schemes, DE intake was increased linearly (P < 0.05) with BW, allowing for an assessment of effects of DE intake and slaughter BW on chemical and physical body composition (carcass, viscera, blood). Between 15 and 90 kg BW, average DE intake of 16.1, 20.9, 25.2, and 28.8 MJ/d supported average BW gains of 502, 731, 899, and 951 g/d, respectively. The proportion of whole-body protein present in the carcass increased with BW and decreased with DE intake (P < 0.05), whereas the distribution of whole-body lipid between carcass and viscera was not influenced by BW and DE intake. A mathematical relationship was developed to determine the relationship between DE intake at slaughter (MJ/d) and chemical body composition in these pigs: whole-body lipid-to-protein ratio = 1.236 - 0.056 x (DE intake) + 0.0013 x (DE intake)2, r2 = 0.71. The data suggests that absolute DE intake alone was an adequate predictor of chemical body composition in this population of entire male pigs over the BW and DE intake ranges that were evaluated, simplifying the characterization of this aspect of nutrition partitioning for growth in different pig populations.

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A unique process has been developed to convert bituminous coal by controlled wet oxidation followed by base treatment to a water-soluble humate called oxihumate. Oxihumate inhibited HIV-1 infection of MT-2 cells with an IC(50) of 12.5 microg/ml. Treatment of free and cell-attached HIV with oxihumate irreversibly reduced infectivity, while the susceptibility of target cells to the virus was not impaired by treatment prior to infection. The infectivity of the HIV particles was inhibited by interference with CD4 binding and the V3 loop-mediated step of virus entry. No viral resistance to oxihumate developed over a 12-week period in vitro. Oxihumate therefore holds promise for the treatment of HIV-infected patients.

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To mediate adaptation to stimuli, the methyltransferase (CheR) catalyzes methyl group transfer from S-adenosyl-L-methionine (SAM) to glutamyl residues in the transmembrane receptors of the bacterial chemosensory signaling pathway. The interaction between receptors and CheR occurs at two sites: a methylation site-active site interaction, and a 'docking' site interaction that is separated both from the methylation sites and the CheR active site. It is not certain if the docking site interaction functions merely to localize the transferase in close proximity to the methylation sites, or if it also increases CheR catalytic activity. Isothermal titration calorimetry experiments are conducted to test for allosteric interactions between the docking and active sites on CheR, which are expected to be present if docking activates CheR. The binding parameters (DeltaG, DeltaH, DeltaS) of a substrate analog of SAM, S-adenosyl-L-homocysteine (SAH), are measured both in the absence and presence of saturating concentrations of a pentapeptide (NWETF) that defines the docking receptor docking segment. SAH binding is unaffected by the presence of saturating NWETF, providing evidence that an allosteric activation of CheR does not take place upon docking, and thus supports the idea that the CheR-NWETF interaction merely functions to localize CheR near the sites of methylation.

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We report a rare case of unilateral agenesis of the internal carotid artery in association with congenital anterior hypopituitarism. The collateral circulation is supplied by a transsellar intercavernous anastomotic vessel connecting the internal carotid arteries. These abnormalities are well depicted on MRI and MRA. The agenesis of the internal carotid artery may explain the pathogenesis of some of congenital anterior hypopituitarism.

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Intensive study of bacterial chemoreceptors has not yet revealed how receptor methylation and ligand binding alter the interactions between the receptor cytoplasmic domain and the CheA kinase to control kinase activity. Both monomeric and dimeric forms of an Asp receptor cytoplasmic fragment have been shown to be highly dynamic, with a small core of slowly exchanging amide hydrogens (Seeley, S. K., Weis, R. M., and Thompson, L. K. (1996) Biochemistry 35, 5199-5206). Hydrogen exchange studies of the wild-type cytoplasmic fragment and an S461L mutant thought to mimic the kinase-inactivating state are used to investigate the relationship between the stable core and dimer dissociation. Our results establish that (i) decreasing pH stabilizes the dimeric state, (ii) the stable core is present also in the transition state for dissociation, and (iii) this core is expanded significantly by small changes in electrostatic and hydrophobic interactions. These kinase-inactivating changes stabilize both the monomeric and the dimeric states of the protein, which has interesting implications for the mechanism of kinase activation. We conclude that the cytoplasmic domain is a flexible region poised for stabilization by small changes in electrostatic and hydrophobic interactions such as those caused by methylation of glutamate residues and by ligand-induced conformational changes during signaling.

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BACKGROUND: Gillespie syndrome is the phenotype partial aniridia, cerebellar ataxia and mental retardation. Further malformations can be associated, mainly females are affected. Inheritance and genetics of the syndrome are unknown. Autosomal dominant aniridia is an important differential diagnosis of fixed dilated pupils and is usually associated by mutations of the PAX6 gene. In 1998 the first report of a chromosomal abnormality presenting a de novo translocation t(X;11) (p22.32;p12) detected in a patient with Gillespie syndrome has been published. PATIENTS AND METHODS: A 8 year-old girl with Gillespie syndrome phenotype associated with congenital pulmonary stenosis and helix dysplasia is reported. Karyotyping as well as molecular biological investigations of the PAX6 gene were performed. RESULTS: The karyotype of the girl and her clinically inconspicuous mother showed no abnormalities, especially no de novo translocation of the chromosomes X and 11. PAX6 gene analysis of the affected girl presented no mutations. CONCLUSIONS: The combination of muscular hypotonia and fixed dilated pupils in infancy is suspicious of Gillespie syndrome. Congenital pulmonary stenosis and helix dysplasia can be associated. PAX6 gene analysis can be helpful to distinguish between autosomal dominant aniridia and Gillespie syndrome. To illucidate the underlying genetic defects karyotyping and the search for de novo translocations especially of chromosome X and 11 should be performed.

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In the Escherichia coli chemosensory pathway, receptor modification mediates adaptation to ligand. Evidence is presented that covalent modification influences ligand binding to receptors in complexes with CheW and the kinase CheA. Kinase inhibition was measured with serine receptor complexes in different modification levels; Ki for serine-mediated inhibition increased 10,000-fold from the lowest to the highest level. Without CheA and CheW, ligand binding is unaffected by covalent modification; thus, the influence of covalent modification is mediated only in the receptor complex, a conclusion supported by an analogy to allosteric enzymes and the observation of cooperative kinase inhibition. Also, the finding that a subsaturating serine concentration accelerates active receptor-kinase complex assembly implies that the assembly/disassembly process may also contribute to kinase regulation.

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We report two children with acute loss of neurological functions and signs of an increased intracranial pressure. Imaging techniques ruled out space occupying lesions, whereas CSF cytology indicated CNS involvement of a non-Hodgkin lymphoma in the form of abnormal lymphocytic pleocytosis with malignancy criteria fulfilling lymphoid cells. CSF protein electrophoresis and Borrelia burgdorferi serology revealed neuroborreliosis which was successfully treated with antibiotic therapy. The malignancy mimicking cytology is based on a blastoid transformation of B- and T-lymphocytes due to the antigenic stimulus of B. burgdorferi infection. Lymphoid cells in the CSF of a patient with acute or chronic neurological symptoms raise the differential diagnosis of inflammatory etiology versus CNS lymphoma. Monomorphism and higher quantity of the lymphoid cells point to CNS lymphoma. A lower quantity and polyclonal pattern of lymphoid cells associated with an elevated protein fraction caused by intrathecal immunoglobulin synthesis suggest an inflammatory etiology.

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Tay's syndrome is a trichothiodystrophy associated with congenital ichthyosis. We report the findings on MRI and spectroscopy in a young girl with sparse, short, ruffled hair, dry skin and delayed milestones. T2-weighted images showed prominent diffuse confluent increase in signal symmetrically in all the supratentorial white matter. These findings are similar to those in a previously described case, and consistent with dysmyelination. Spectroscopy showed increased myoinositol and decreased choline.

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The intergeniculate leaflet (IGL) and the ventral lateral geniculate nucleus (VLG) are ventral thalamic derivatives within the lateral geniculate complex. In this study, IGL and VLG efferent projections were compared by using anterograde transport of Phaseolus vulgaris-leucoagglutinin and retrograde transport of FluoroGold. Projections from the IGL and VLG leave the geniculate in four pathways. A dorsal pathway innervates the thalamic lateral dorsal nucleus (VLG), the reuniens and rhomboid nuclei (VLG and IGL), and the paraventricular nucleus (IGL). A ventral pathway runs through the geniculohypothalamic tract to the suprachiasmatic nucleus and the anterior hypothalamus (IGL). A medial pathway innervates the zona incerta and dorsal hypothalamus (VLG and IGL); the lateral hypothalamus and perifornical area (VLG); and the retrochiasmatic area (RCA), dorsomedial hypothalamic nucleus, and subparaventricular zone (IGL). A caudal pathway projects medially to the posterior hypothalamic area and periaqueductal gray and caudally along the brachium of the superior colliculus to the medial pretectal area and the nucleus of the optic tract (IGL and VLG). Caudal IGL axons also terminate in the olivary pretectal nucleus, the superficial gray of the superior colliculus, and the lateral and dorsal terminal nuclei of the accessory optic system. Caudal VLG projections innervate the lateral posterior nucleus, the anterior pretectal nucleus, the intermediate and deep gray of the superior colliculus, the dorsal terminal nucleus, the midbrain lateral tegmental field, the interpeduncular nucleus, the ventral pontine reticular formation, the medial and lateral pontine gray, the parabrachial region, and the accessory inferior olive. This pattern of IGL and VLG projections is consistent with our understanding of the distinct functions of each of these ventral thalamic derivatives.

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BACKGROUND: The emergence of bacteria that are resistant to vancomycin (V), a glycopeptide antibiotic, results from the replacement of the carboxy-terminal D-Ala-D-Ala of bacterial cell wall precursors by D-Ala-D-lactate. Recently, it has been demonstrated that covalent dimeric variants of V are active against vancomycin-resistant enterococci (VRE). To study the contribution of divalency to the activities of these variants, we modeled the interactions of V and a dimeric V with L-Lys-D-Ala-D-lactate, an analog of the cell-wall precursors of the vancomycin-resistant bacteria. RESULTS: A dimeric derivative of V (V-Rd-V) was found to be much more effective than V in inhibiting the growth of VRE. The interactions of V and V-Rd-V with a monomeric lactate ligand - diacetyl-L-Lys-D-Ala-D-lactate (Ac2KDADLac) - and a dimeric derivative of L-Lys-D-Ala-D-lactate (Lac-R'd-Lac) in solution have been examined using isothermal titration calorimetry and UV spectroscopy titrations; the results reveal that V-Rd-V binds Lac-R'd-Lac approximately 40 times more tightly than V binds Ac2KDADLac. Binding of V and of V-Rd-V to Nalpha-Ac-L-Lys-D-Ala-D-lactate presented on the surface of mixed self-assembled monolayers (SAMs) of alkanethiolates on gold indicates that the apparent off-rate for dissociation of V-Rd-V from the surface is much slower than that of V from the same surface. CONCLUSIONS: The results are compatible with the hypothesis that divalency is responsible for tight binding, which correlates with small values of minimum inhibitory concentrations of V and V-Rd-V.

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The interaction of simian foamy viruses (FVs) with their putative cellular receptor(s) was studied with two types of recombinant envelope protein (Env). Transient expression of full-length Env in BHK-21 cells induced syncytia formation. However, selected stable transfectants fused with naive cells but not with each other. A soluble fusion protein of the Env surface domain with the Fc fragment of a human IgG1 heavy chain (EnvSU-Ig) was produced in the baculovirus expression system, purified to homogeneity, and used for binding and competition analyses. EnvSU-Ig but not unrelated Ig fusion proteins bound to cells specifically. Neutralizing serum blocked binding of EnvSU-Ig and, vice versa, serum-mediated neutralization was abrogated by the chimeric protein. Concomitant reduction of EnvSU-Ig binding and FV susceptibility was seen in Env-expressing target cells. Although EnvSU-Ig did not inhibit FV infection, very likely due to its displacement by multivalent virus-cell interactions, this divalent ligand should help to characterize functionally and to identify the ubiquitous FV receptor.

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The emphasis our society places on physical fitness has produced an ever increasing number of injuries to the anterior cruciate ligament (ACL). To successfully replace the anterior cruciate deficient knee, physicians need to determine the "ideal" location for the femoral tunnel. In the search to find this "isometric point," if there is one, we draw attention to the origin of the distal fascicles of the anterior cruciate from the lateral femoral condyle. Visualizing this surgical landmark can facilitate and expedite accurate placement of the isometric device and thus influence the results of the surgical procedure. Also, magnetic resonance imaging may help the surgeon in locating the origin of the ACL.

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Tris(vancomycin carboxamide) binds a trivalent ligand derived from D-Ala-D-Ala with very high affinity: dissociation constant (Kd) approximately 4 x 10(-17) +/- 1 x 10(-17) M. High-affinity trivalent binding and monovalent binding are fundamentally different. In trivalent (and more generally, polyvalent) binding, dissociation occurs in stages, and its rate can be accelerated by monovalent ligand at sufficiently high concentrations. In monovalent binding, dissociation is determined solely by the rate constant for dissociation and cannot be accelerated by added monomer. Calorimetric measurements for the trivalent system indicate an approximately additive gain in enthalpy relative to the corresponding monomers. This system is one of the most stable organic receptor-ligand pairs involving small molecules that is known. It illustrates the practicality of designing very high-affinity systems based on polyvalency.

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