Non-covalent molecular imprinting with emphasis on its application in separation and drug development

The molecular imprinting technique can be defined as the formation of specific nano-sized cavities by means of template-directed synthesis. The resulting molecularly imprinted polymers (MIPs), which often have an affinity and a selectivity approaching those of antibody-antigen systems, have thus been coined "artificial antibodies." MIPs are characterized by their high specificity, ease of preparation, and their thermal and chemical stability. They have been widely studied in connection with many potential applications, including their use for separation and isolation purposes, as antibody mimics (biomimetic assays and sensors), as enzyme mimics, in organic synthesis, and in drug delivery. The non-covalent imprinting approach, developed mainly in Lund, has proven to be more versatile than the alternative covalent approach because of its preparation being less complicated and of the broad selection of functional monomers and possible target molecules that are available. The paper presents a review of studies of this versatile technique in the areas of separation and drug development, with emphasis being placed on work carried out in our laboratory.     

Significant sequence similarities in promoters and precursors of Arabidopsis thaliana non-conserved microRNAs

Some plant microRNAs have been shown to be de novo generated by inverted duplication from their target genes. Subsequent duplication events potentially generate multigene microRNA families. Within this article we provide supportive evidence for the inverted duplication model of plant microRNA evolution. First, we report that the precursors of four Arabidopsis thaliana microRNA families, miR157, miR158, miR405 and miR447 share nearly identical nucleotide sequences throughout the whole miRNA precursor between the family members. The extent and degree of sequence conservation is suggestive of recent evolutionary duplication events. Furthermore we found that sequence similarities are not restricted to the transcribed part but extend into the promoter regions. Thus the duplication event most probably included the promoter regions as well. Conserved elements in upstream regions of miR163 and its targets were also detected. This implies that the inverted duplication of target genes, at least in certain cases, had included the promoters of the target genes. Sequence conservation within promoters of miRNA families as well as between miRNA and its potential progenitor gene can be exploited for understanding the regulation of microRNA genes.     

Simulation of Rapoport's rule for latitudinal species spread

Rapoport's rule claims that latitudinal ranges of plant and animal species are generally smaller at low than at high latitudes. However, doubts as to the generality of the rule have been expressed, because studies providing evidence against the rule are more numerous than those in support of it. In groups for which support has been provided, the trend of increasing latitudinal ranges with latitude is restricted to or at least most distinct at high latitudes, suggesting that the effect may be a local phenomenon, for example the result of glaciations. Here we test the rule using two models, a simple one-dimensional one with a fixed number of animals expanding in a northern or southerly direction only, and the evolutionary/ecological Chowdhury model using birth, ageing, death, mutation, speciation, prey-predator relations and food levels. Simulations with both models gave results contradicting Rapoport's rule. In the first, latitudinal ranges were roughly independent of latitude, in the second, latitudinal ranges were greatest at low latitudes, as also shown empirically for some well-studied groups of animals.     

Antibiotic efficacy and microbial virulence during space flight

Human space flight is a complex undertaking that entails numerous technological and biomedical challenges. Engineers and scientists endeavor, to the extent possible, to identify and mitigate the ensuing risks. The potential for an outbreak of an infectious disease in a spacecraft presents one such concern, which is compounded by several components unique to an extraterrestrial environment. Various factors associated with the space flight environment have been shown to potentially compromise the immune system of astronauts, increase microbial proliferation and microflora exchange, alter virulence and decrease antibiotic effectiveness. An acceptable resolution of the above concerns must be achieved to ensure safe and efficient space habitation. To help bring this about, scientists are employing advances in biotechnology to better characterize the relevant variables and establish appropriate solutions. Because many of these clinical concerns are also relevant in terrestrial society, this research will have reciprocal benefits back on Earth.     

Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56

Myxobacteria belonging to the genus Sorangium are known to produce a variety of biologically active secondary metabolites. Chivosazol is a macrocyclic antibiotic active against yeast, filamentous fungi and especially against mammalian cells. The compound specifically destroys the actin skeleton of eucaryotic cells and does not show activity against bacteria. Chivosazol contains an oxazole ring and a glycosidically bound 6-deoxyglucose (except for chivosazol F). In this paper we describe the biosynthetic gene cluster that directs chivosazol biosynthesis in the model strain Sorangium cellulosum So ce56. This biosynthetic gene cluster spans 92 kbp on the chromosome and contains four polyketide synthase genes and one hybrid polyketide synthase/nonribosomal peptide synthetase gene. An additional gene encoding a protein with similarity to different methyltransferases and presumably involved in post-polyketide modification was identified downstream of the core biosynthetic gene cluster. The chivosazol biosynthetic gene locus belongs to the recently identified and rapidly growing class of trans-acyltransferase polyketide synthases, which do not contain acyltransferase domains integrated into the multimodular megasynthetases.     

Keratin 5 knockout mice reveal plasticity of keratin expression in the corneal epithelium

We have recently demonstrated that the keratin K3 gene, which is active in the suprabasal human corneal epithelium, is missing in the genome of the mouse. We show that a normal K3 gene exists in a wide variety of mammals while in rodents the gene is converted to a pseudogene with a very strong sequence drift. The availability of K5-/- mice provides a unique opportunity to investigate type-specific keratin function during corneal differentiation in the absence of both K5 and K3. Here, we report that the deletion of K5, which in wild-type mice forms a cytoskeleton with K12, does neither cause keratin aggregation nor cytolysis in the cornea. This is due to the induction of K4 in corneal epithelial cells, normally restricted to corneal stem stem cells residing in the limbus. Using a combination of antibodies and RT-PCR, we identified additional keratins expressed in the mouse cornea including K23 which was previously thought to be specific for pancreatic carcinomas. This reflects an unexpected complexity of keratin expression in the cornea. Our data suggest that in the absence of mechanical stress, corneal differentiation does not depend on distinct keratin pairs, supporting a concept of functional redundancy, at least for certain keratins.     

Convergence of sensory pathways in the development of somatic and visceral hypersensitivity

Sensory neurons innervating different tissues converge onto second-order neurons in the spinal cord. We examined whether inflammation or transient overexpression of nerve growth factor (NGF) in one tissue triggers hypersensitivity in referral sites. Thresholds to mechanical and thermal stimulation of the hindpaw, visceromotor responses to colorectal distension, and cystometrograms were performed in appropriate controls and mice with experimentally induced cystitis, inflammation of the hindpaw or front paw, or injection of viral vectors encoding NGF or green fluorescent protein (GFP). Cystitis and NGF but not GFP overexpression in the bladder triggered bladder hyperactivity associated with mechanical and thermal hypersensitivity in cutaneous referral sites and enhanced responses to colorectal distension. Hindpaw inflammation and injection of the NGF- but not GFP-encoding viral vector or front paw inflammation induced mechanical and thermal hyperalgesia in the affected hindpaw and increased responses to colorectal distension without altering the micturition reflex. In conclusion, sensitization of sensory pathways by inflammation or NGF contributes to the development of hypersensitivity in neighboring organs and cutaneous referral sites and provides a potential mechanism underlying the coexistence of pain syndromes in patients with functional diseases.     

Normal T-cell development and immune functions in TRIM-deficient mice

The transmembrane adaptor molecule TRIM is strongly expressed within thymus and in peripheral CD4(+) T cells. Previous studies suggested that TRIM is an integral component of the T-cell receptor (TCR)/CD3 complex and might be involved in regulating TCR cycling. To elucidate the in vivo function of TRIM, we generated TRIM-deficient mice by homologous recombination. TRIM(-/-) mice develop normally and are healthy and fertile. However, the animals show a mild reduction in body weight that appears to be due to a decrease in the size and/or cellularity of many organs. The morphology and anatomy of nonlymphoid as well as primary and secondary lymphoid organs is normal. The frequency of thymocyte and peripheral T-cell subsets does not differ from control littermates. In addition, a detailed analysis of lymphocyte development revealed that TRIM is not required for either positive or negative selection. Although TRIM(-/-) CD4(+) T cells showed an augmented phosphorylation of the serine/threonine kinase Akt, the in vitro characterization of peripheral T cells indicated that proliferation, survival, activation-induced cell death, migration, adhesion, TCR internalization and recycling, TCR-mediated calcium fluxes, tyrosine phosphorylation, and mitogen-activated protein family kinase activation are not affected in the absence of TRIM. Similarly, the in vivo immune response to T-dependent and T-independent antigens as well as the clinical course of experimental autoimmune encephalomyelitis, a complex Th1-mediated autoimmune model, is comparable to that of wild-type animals. Collectively, these results demonstrate that TRIM is dispensable for T-cell development and peripheral immune functions. The lack of an evident phenotype could indicate that TRIM shares redundant functions with other transmembrane adaptors involved in regulating the immune response.