Temperature-Switchable Control of Ligand Display on Adlayers of Mixed Poly(lysine)-g-(PEO) and Poly(lysine)-g-(ligand-modified poly-N-isopropylacrylamide).

Adlayers of poly(lysine)-g-PEG comblike copolymer are extensively used to prepare cell-repellant and protein-repellent surfaces by a straightforward coulomb-driven adsorption that is compatible with diverse substrates (glass, Petri dish, etc.). To endow surfaces with functional properties, namely, controlled ligand-protein binding, comblike poly(lysine) derivatives were used to deposit temperature-responsive poly(NIPAM) macrografts mixed with PEG ones on glass surfaces. Simple surface immersion in mixed solutions of biotin-modified poly(lysine)-g-poly(N-isopropylacrylamide) and poly(lysine)-g-poly(ethylene oxide) yielded robust adlayers whose composition reflected the ratio between the two polymers in solution. We show by fluorescence imaging, and comparison with repellent 100% PEGylated patterns, that specific binding of model avidin/particle conjugates (diameters of ca. 10 or 200 nm) was controlled by temperature switch. The biotin ligand was displayed and accessible at low T, or hidden at T > LCST. Topography and mechanical mapping measurements by AFM confirmed the swelling/collapse status of PNIPAM macrografts in the adlayer at low/high T, respectively. Temperature-responsive comblike PLL derivative that can spontaneously cover anionic interfaces is a promising platform enabling good control on the deposition and accessibility of biofunctional groups on various solid surfaces.

Increased cardiac mRNA expression of matrix metalloproteinase-1 (MMP-1) and its inhibitor (TIMP-1) in DCM patients.

Left ventricular dilation and myocardial remodeling are hallmarks of dilated cardiomyopathy (DCM). It is assumed that left ventricular dilation is caused by the disintegration of the collagenous network by increased collagenolytic activity of matrix metalloproteinases (MMPs) and their adequate tissue inhibitors (TIMPs). In this study the myocardial MMP-1 and TIMP-1 mRNA expressions were investigated by using real-time quantitative PCR analysis from right septal endomyocardial biopsies of patients with dilated cardiomyopathy (n = 46) and control subjects (n = 11). The volume density (Vv%) of collagen was measured morphometrically. Classification was done according to LV diameters [left ventricular enddiastolic diameter (LVEDD, cm) calculated to body surface area (BSA, m(2))] into three DCM groups: group I (LVEDD-BSA > 2.7-3.0 cm/m(2)), group II ( > 3.0-3.6 cm/m(2)), group III ( > 3.6 cm/m(2)), controls (< 2.7 cm/m(2)). Compared with controls, the MMP-1 expression in patients with DCM was significantly increased (119.2 +/- 45.2 vs. 1.3 +/- 0.4; p < 0.001) as was TIMP-1 expression (9.6 +/- 1.2 vs. 1.3 +/- 0.4; p < 0.01). Moreover the MMP-1 and TIMP-1 expression varied according to LV diameter: group I (MMP-1: 8.7 +/- 3.5; p = 0.33; TIMP- 1: 4.5 +/- 1.2; p < 0.01); group II (MMP-1: 211.4 +/- 86.0; p < 0.001; TIMP-1: 12.5 +/- 1.9 ; p < 0.001); group III (MMP-1: 38.8 +/- 22.6; p < 0.01; TIMP-1: 8.1 +/- 1.7; p < 0.001). Compared with controls, the collagen level in DCMPt. was significantly increased: 5.0 +/- 0.6 vol% vs 1.2 +/- 0.2 vol% p < 0.001 and correlates with LV diameter. This study reveals that the overexpression of MMP-1, which is associated with an increased ratio of MMP-1/TIMP-1 in DCM, indicates an activated collagenolytic system while replacement fibrosis is accumulating. The MMP-1 overexpression is mainly found in moderately dilated DCM hearts (group II) indicating the dynamic process of LV dilation and the importance of collagenases in the early phase of LV remodeling.

Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins.

Electron crystallography and atomic force microscopy allow the study of two-dimensional membrane protein crystals. While electron crystallography provides atomic scale three-dimensional density maps, atomic force microscopy gives insight into the surface structure and dynamics at sub-nanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed. In this way, the function-related conformational changes can be assessed, thus providing a detailed insight on the molecular mechanisms of essential biological processes.

Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking.

The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPases associated with diverse cellular activities) and a key player in the transport of proteins out of a prevacuolar endosomal compartment. In human cells, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibit a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS4-A, to a partial and, in the case of human VPS4-B, to a complete suppression of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterologous expressed human VPS4-B protein, and partially by the human VPS4-A protein. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced dominant-negative vacuolar protein sorting defects in wild-type yeast cells in both cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B proteins can form heteromeric complexes, and subcellular localisation experiments indicate that both human VPS4 proteins associate with endosomal compartments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast.

High-resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2.

Light-harvesting complexes 2 (LH2) are the accessory antenna proteins in the bacterial photosynthetic apparatus and are built up of alphabeta-heterodimers containing three bacteriochlorophylls and one carotenoid each. We have used atomic force microscopy (AFM) to investigate reconstituted LH2 from Rubrivivax gelatinosus, which has a C-terminal hydrophobic extension of 21 amino acids on the alpha-subunit. High-resolution topographs revealed a nonameric organization of the regularly packed cylindrical complexes incorporated into the membrane in both orientations. Native LH2 showed one surface which protruded by approximately 6 A and one that protruded by approximately 14 A from the membrane. Topographs of samples reconstituted with thermolysin-digested LH2 revealed a height reduction of the strongly protruding surface to approximately 9 A, and a change of its surface appearance. These results suggested that the alpha-subunit of R.gelatinosus comprises a single transmembrane helix and an extrinsic C-terminus, and allowed the periplasmic surface to be assigned. Occasionally, large rings ( approximately 120 A diameter) surrounded by LH2 rings were observed. Their diameter and appearance suggest the large rings to be LH1 complexes.

Sequence, organization, chromosomal localization, and alternative splicing of the human serine protease inhibitor gene hurpin (PI13) which is upregulated in psoriasis.

Hurpin (protease inhibitor 13; PI13) is the most recently identified member of the ovalbumin family of serine protease inhibitors (serpins). It is expressed in human epidermal keratinocytes and is downregulated by exposure to ultraviolet irradiation. A role for hurpin in the proliferation or differentiation of keratinocytes has been proposed because of its strong expression in proliferating cells and its deregulated expression in the lesional epidermis of psoriatic patients. Here, we report the cloning, chromosomal localization, and complete sequence of the human hurpin gene. By PCR-based screening of the GeneBridge 4 radiation hybrid panel, we mapped the gene to chromosome 18q21.3, close to a known cluster of ov-serpin genes. Using the full-length cDNA for hurpin, we identified two clones from an arrayed genomic P1 placental library that contain the entire hurpin gene. Sequencing revealed that the gene covers 12.253 kb and is comprised of eight exons and seven introns. The exon--intron boundaries are identical in position and phasing to those in other members of the 18q serpin gene cluster, and analysis of hurpin variants indicated that modified functional inhibitors, differing only in the CD interhelical loop, can be generated by differential splicing of exon 3. These data show that hurpin is a typical member of the 18q ovalbumin-serpins most closely related to the serpins squamous-cell carcinoma antigens 1 and 2.

In vitro development of resistance to six quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus.

Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.

Development of resistance to ciprofloxacin, rifampin, and mupirocin in methicillin-susceptible and -resistant Staphylococcus aureus isolates.

A relationship between resistance to methicillin and resistance to fluoroquinolones, rifampin, and mupirocin has been described for Staphylococcus aureus. Differences in resistance rates may be explainable by a higher spontaneous mutation rate (MR) or a faster development of resistance (DIFF) in methicillin-resistant S. aureus (MRSA). No differences in MR, DIFF, and mutations in grlA and gyrA were detected between methicillin-susceptible S. aureus and MRSA. The higher resistance rates in MRSA are not the result of hypermutability of target genes or a faster emergence of different mutations and may be the consequence of clonal spread of multiresistant MRSA.

Complete sequencing and mRNA expression analysis of the MEN-I gene in adrenal myelolipoma.

The molecular pathogenesis of adrenal myelolipoma is unclear. Endocrine activity of these tumors and association with other endocrine tumors have stimulated the hypothesis that it may belong to the group of sporadic tumors caused by defects of the gene responsible for multiple endocrine neoplasia type I (MEN-I). DNA of blood and tumoral sections from two patients with adrenal myelolipoma were analyzed by examination of variable number of tandem repeats (VNTR) loci PYGM, D11S987, D11S480, and D11S449 on chromosome 11q13 and by complete direct DNA sequencing of all coding exons and splice junctions of the MEN-I gene. Menin expression was examined by RT-PCR. RT-PCR did not detect menin expression in one adrenal myelolipoma. No loss of heterozygozity on chromosome 11q13 was identified. Intragenic heterozygozity was retained in codon 418 of the menin gene in both patients. No mutation was identified in the coding exons of the menin gene. Complete DNA sequencing yielded no hint that defects of the MEN-I gene are responsible for the formation of adrenal myelolipomas. Adrenal myelolipomas do not share the loss of heterozygozity on chromosome 11q13 observed in some benign adenomatous and many malignant adrenocortical tumors.

The aquaporin sidedness revisited.

Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of micro-organisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore. Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains oriented at 180 degrees to each other poses a significant challenge in the determination of sidedness. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy. The shadowing of some surfaces was prevented by protruding aggregates. Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal. Cross-correlation peak searches along lattices crossing metal-coated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps. AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 A three-dimensional structure of AQP1. We conclude that the sidedness we originally proposed for aquaporin density maps was inverted.

Sequence analysis of the MEN I gene in two patients with multiple cutaneous lipomas and endocrine tumors.

The discovery of mutations of the menin gene in a few multiple endocrine neoplasma type 1 (MEN I)-associated lipomas and loss of heterozygosity (LOH) on chromosome 11q13 in some sporadic lipomas has stimulated the hypothesis that lipomas may belong to the group of sporadic tumors caused by defects of the gene responsible for MEN I. Since it is unclear if the above hypothesis applies to all patients with lipoma or just to specific subsets, we searched to enlarge the database on this topic. For this purpose, we identified two patients with multiple cutaneous lipomas. One had an additional pituitary adenoma and familial presentation of multiple lipomas, the other had recurrent goiter in the setting of a family history of adenomatous goiter. Deoxyribonucleic acid (DNA) was analyzed by complete direct DNA sequencing of all coding exons and splice junctions of the MEN I gene. No mutation was identified in the coding exons of the menin gene. In contrast to former data on sporadic lipomas, these data are the first to render evidence that mutations of the MEN I gene may not be responsible for the formation of multiple lipomas, even if they appear in the context of other endocrine tumors.

Complete sequencing and messenger ribonucleic acid expression analysis of the MEN I gene in adrenal cancer.

Adrenal cancer is a rare sporadic disease that has also been observed in the context of multiple endocrine neoplasia type I (MEN I). Adrenal lesions occur in up to 40% of MEN I patients. Loss of heterozygosity of the 11q13 band harboring the menin gene has been reported in more than 50% of patients with adrenal cancer. Despite this high index of suspicion, former screening studies did not reveal mutations of the MEN I gene in 28 patients. We identified loss of heterozygosity of 11q13 microsatellites in five of five patients (100%). In 40%, heterozygosity was retained in codon 418 of the MEN I gene. Complete direct DNA sequencing data of the entire coding region and adjacent splice sites of the MEN I gene were obtained in 14 patients with sporadic adrenal cancer. In only one of them a heterozygous missense mutation, R176Q (exon 3), was identified. Due to the heterozygous pattern and unknown biological effect of this mutation, it is not clear whether there is a causal relationship with adrenal cancer. The total mutation frequency in sporadic adrenal cancer is 1 of 14 (7%). Menin messenger RNA expression was identified in 14 of 14 patients (100%). Transcriptional inactivation of the menin gene is, hence, unlikely to cause loss of its tumor suppressor function in adrenal cancer. Furthermore, we examined three patients who presented adrenal cancer in the context of sporadic multiglandular endocrine tumor disease previously diagnosed on clinical grounds to be MEN I syndrome. An opal stop codon mutation was identified in codon 126 (exon 2) in the adrenal cancer of one of these patients. Formation of the adrenal cancer in this patient may be rather coincidental because the mutation was present in a heterozygous pattern. There was no mutation of the menin gene in the two other patients. This may mean that formation of adrenal cancer in the context of multiglandular endocrine disease denotes an entity different from MEN I in some patients.

Prevalence of gyrA, gyrB, parC, and parE mutations in clinical isolates of Streptococcus pneumoniae with decreased susceptibilities to different fluoroquinolones and originating from Worldwide Surveillance Studies during the 1997-1998 respiratory season.

From 8,419 worldwide clinical isolates of Streptococcus pneumoniae obtained during 1997-1998, 69 isolates with reduced susceptibility or resistance to fluoroquinolones (FQs) were molecularly characterized. For the isolates in this prevalence study, only parC (Ser-79-->Tyr) and gyrA (Ser-81-->Phe or Tyr) mutations, especially in combination, were found to contribute significantly to resistance. These mutations influenced the FQ MICs to varying degrees, although the rank order of activity remains independent of mutation type, with ciprofloxacin the least active, followed by levofloxacin, gatifloxacin/grepafloxacin/moxifloxacin/sparfloxaci n/trovafloxacin, and clinafloxacin/sitafloxacin. Efflux likely plays a crucial role in reduced susceptibility for new hydrophilic FQs.

Association of alterations in ParC and GyrA proteins with resistance of clinical isolates of Enterococcus faecium to nine different fluoroquinolones.

The parC and gyrA genes of 73 ciprofloxacin-resistant and 6 ciprofloxacin-susceptible Enterococcus faecium clinical isolates were partly sequenced. Alterations in ParC and GyrA, possibly in combination with other resistance mechanisms, severely restricted the in vitro activities of the nine quinolones tested. For all isolates, clinafloxacin and sitafloxacin showed the best activities.

High resolution AFM topographs of the Escherichia coli water channel aquaporin Z.

Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning alpha-helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p42(1)2 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change.

Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes clinical isolates with alterations in GyrA and ParC proteins.

The in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes. Irrespective of the alterations in GyrA and ParC proteins, clinafloxacin exhibited greater activity than all other fluoroquinolones tested against K. pneumoniae and E. aerogenes.

Cloning, characterisation, and functional expression of the Mus musculus SKD1 gene in yeast demonstrates that the mouse SKD1 and the yeast VPS4 genes are orthologues and involved in intracellular protein trafficking.

The mouse SKD1 protein displays a high degree of sequence identity (62%) to the yeast Vps4 protein, which is involved in the transport of proteins out of a prevacuolar/endosomal compartment. We isolated the mouse SKD1 locus and found that the SKD1 gene is split into 11 exons covering a region of 29kb of the genome. Interestingly, the exon/intron structure reflects to a certain degree the proposed domain structure of the protein, since the 5' located coiled-coil region and the AAA domain are flanked by introns. Analysis of the promoter region, which revealed features common for 'housekeeping genes', is consistent with previous results of a mouse multi-tissue Northern blot, confirming that SKD1 is a ubiquitously expressed gene. Expression of the full-length SKD1 cDNA in a vps4 disrupted yeast strain suppressed the temperature-sensitive growth defect of the vps4 mutant strain. Overexpression of wild type and expression of mutant Vps4 and SKD1 proteins, harbouring single amino acid exchanges in their AAA domains, induced a dominant-negative vacuolar protein sorting defect in wild type yeast cells, indicating that mouse SKD1 protein and yeast Vps4p fulfil similar functions.