Laser desorption/ionization—Mass spectrometry using mesoporous silicate as matrix for the analysis of various molecules

In this study, mesoporous silicate was applied as a matrix for the analysis of various molecules from small molecules to medium sized peptides in laser desorption/ionization mass spectrometry. In contrast with conventional matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), the proposed approach desorption/ionization on mesoporous silicate mass spectrometry (DIOM-MS), significantly reduces the problem of matrix interference in low mass region and can be applied to the analysis of versatile chemicals including amino acids, synthetic drugs, peptides and others. In addition, distinctive advantage of DIOM-MS showed higher salt tolerance and could be applied to identify the proteins from the analysis of tryptically digested peptides. DIOM-MS has several availabilities such as easy sample preparation, rapid analysis of small molecules without noise, peptide analysis without organic matrix, high salt tolerance, versatile coupling with other separation techniques, and high throughput manner.     

Cytogenetic analysis of the 2B3-4 — 2B11 region of the X chromosome of Drosophila melanogaster

Puffing patterns have been studied both in homozygotes t10/t10, a gene located in the area of the early ecdysone puff 2B5, and in a yellow (y) control stock, at the end of the third instar and during prepupal development. In mutants t10 at the end of the third instar puffing develops normally in general, however, 21 puffs (5 early and 16 late ones) underdevelop or do not develop at all, some larval intermoult puffs regressing slower. The next cycle of puffs (mid prepupal) in mutants t10 proceeds normally, but in the late prepupal cycle 21 puffs underdevelop again or are not formed at all. A model for the induction of early ecdysone puffs is proposed, assigning a key role to the 2B5 puff product in stimulating other early puffs. It is suggested that defects in the activity of early puffs in the mutant t10 may cause underdevelopment of late puffs.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Infrared and raman study of matrix isolated M(SO2) molecules. The structure of the molecular ion SO2−

The IR and Raman spectra of Cs(SO₂), K(SO₂) and Na(SO₂) molecules were studied by ³²S/³⁴S and ¹⁶O/¹⁸O isotopic substitution technique. These molecules have a planar ring configuration of C_2v symmetry with the OSO angle equal to 109°±5° and the SO bond length of 0.149±0.001 nm. The alkali metal atom interacts symmetrically with the oxygen atoms of the SO₂⁻ group. The doubling observed for the vibrations of Cs(S¹⁶O¹⁸O) was attributed to a matrix effect.     

Detection of dynamic water molecules in a microcrystalline sample of the SH3 domain of α-spectrin by MAS solid-state NMR

Water molecules are a major determinant of protein stability and are important for understanding protein–protein interactions. We present two experiments which allow to measure first the effective T₂ decay rate of individual amide proton, and second the magnetization build-up rates for a selective transfer from H₂O to H^N using spin diffusion as a mixing element. The experiments are demonstrated for a uniformly ²H, ¹⁵N labeled sample of a microcrystalline SH3 domain in which exchangeable deuterons were back-substituted with protons. In order to evaluate the NMR experimental data, as X-ray structure of the protein was determined using the same crystallization protocol as for the solid-state NMR sample. The NMR experimental data are correlated with the dipolar couplings calculated from H₂O–H^N distances which were extracted from the X-ray structure of the protein. We find that the H^N T₂ decay rates and H₂O–H^N build-up rates are sensitive to distance and dynamics of the detected water molecules with respect to the protein. We show that qualitative information about localization and dynamics of internal water molecules can be obtained in the solid-state by interpretation of the spin dynamics of a reporter amide proton.     

On the properties of Se ⋯N interaction: the analysis of substituent effects by energy decomposition and orbital interaction

The nature and strength of intermolecular Se ?N interaction between selenium-containing compounds HSeX (X = CH₃, NH₂, CF₃, OCH₃, CN, OH, NO₂, Cl, F), and NH₃ have been investigated at the MP2/aug-cc-pVDZ level. The Se ?N interaction is found to be dependent on the substituent groups, which greatly affect the positive electrostatic potential of Se atoms and the accepting electron ability of X-Se σ ^? antibonding orbital. Energy decomposition of the Se ?N interaction reveals that electrostatic and induction forces are comparable in the weak-bonded complexes and induction becomes more significant in the complexes with strong electron-withdrawing substituents. Natural bond orbital (NBO) analysis indicates that the primary source of the induction is the electron transfer from the N lone pair to the X-Se σ ^? antibonding orbital. The geometry of the complex and the interaction directionality of NH₃ to X-Se bond can be regarded as a consequence of the exchange-repulsion. The topological analysis on the electron density reveals the nature of closed-shell interaction in these X-Se ?N contacts. The Se ?N interaction in the complexes with the strong electron-withdrawing substituent has a partly covalent character.     

Sequence analysis of the upstream regions of Xenopus laevis β-globin genes and arrangement of repetitive elements within the globin gene clusters

The globin gene clusters of Xenopus laevis are interspersed by various different repetitive DNA elements. A specific repeat, the JH12 element, has been mapped by Southern analysis and some of its locations have been subsequently confirmed by nucleotide sequencing. JH12 family members seem to represent mobile genetic elements and display a high degree of divergence. The nucleotide sequences upstream to the adult _I-globin gene and to the two coordinately expressed larval _I- and _II genes have been determined and compared to those of the adult -genes. Besides some repetitive DNA elements and a short sequence of rather weak homology we have found no characteristic sequence motifs to be common to the adult - and -genes. The two larval -genes share one short sequence element being absent from the adult genes. This might reflect completely different sequence requirements for protein interactions and for the regulation of adult and larval globin gene expression.     

Ultrastructural analysis of the granulosa — Luteal cell transition in the ovary of the dog

The transition from ovarian granulosa to lutein cell during the estrus cycle of 60 pregnant and non-pregnant beagle bitches was analyzed by light and electron microscopy (both 100 and 1000 KV). Early proestrus was characterized by a gradual rise in serum estrogen levels, hyperplasia of the granulosa cells, the accumulation of follicular fluid, and the development of tortuous intercellular channels. During the second half of proestrus, serum estrogen levels continued to rise, but growth, division, and differentiation of the granulosa cells was minimal. Estrus was marked by the first acceptance of the male and a well-defined LH peak In the subsequent 24 hour period, the granulosa-lutein cells hypertrophy rapidly and develop a large Golgi apparatus, small profiles of granular endoplasmic reticulum, numerous microfilaments, and large gap junctions between the cells. Mitochondria also proliferate, enlarge, and elongate, but retain lamelliform cristae. Luteinization of the cells and progesterone secretion begin just after ovulation which in turn occurs about 24 hours after the LH peak. On the third and fourth day of estrus, numerous small vesicles of agranular endoplasmic reticulum fill the extoplasm and the mitochondria swell up and round off. The vesicles rapidly fuse into whorled and flattened cisternae or anastomosing tubules of agranular endoplasmic reticulum, while the mitochondria develop tubulovesicular cristae. These structures gradually become organized with respect to the basal lamina. The Golgi apparatus is centered over the pole of the nucleus that faces the pericapillary space. Stacked and whorled cisternae of agranular ER develop in the lateral margins and avascular end of the cell while mitochondria and tubular elements of agranular ER predominate in the central medial and most basal portions of the cytoplasm. Microfilaments are ubiquitous and appear to be instrumental in this orientation process. The cell surface develops three distinct regional specializations that coincide with the underlying cellular compartments: interconnecting pleomorphic folds fill the pericapillary space; long tenous microvilli project from the lateral cell surface and form tortuous intercellular channels and canaliculi; and large gap junctions form along the margins of the cell furthest removed from the basal lamina. By the sixth day of estrus, the granulosa-luteal cell transition is nearly complete and serum progesterone levels are on the rise.     

Detection of pro-apoptotic Bax∆2 proteins in the human cerebellum

Bax?2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, Bax?2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of Bax?2 expression in human tissues, we examined a panel of human brain samples. Here, we report four cerebellar cases in which the subjects had no neurological disorder or disease documented. We found Bax?2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong Bax?2-positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of Bax?2 is consistent with that known for Baxα, but in a less ubiquitous manner, these results may imply a potential function of Bax?2 in Purkinje cells.     

Granulocyte adhesion molecules — structure/functionrelationships

Neutrophil responses are regulated by cellular adhesion events, including interaction with. extracellular matrix and other cell types. The diversity of molecular structures which are included in the repertoire of cell adhesion molecules expressed by neutrophils and their subtle regulation allow fine tuning of cell adhesion processes to suit environmental demands. This article reviews some of the recent findings using biochemical, immunochemical and molecular techniques that allow the relationship between adhesion molecule structure and function to be examined. Understanding the molecular basis of cell adhesion events will allow development of novel strategies that allow manipulation of adhesion processes in a clinical setting.     

Phylogenetic analysis of the swine leukocyte antigen — 2 gene for Korean native pigs

The objective of this study was to investigate genetic relationships of the SLA-2 gene, to characterize SLA-2 alleles, and to provide basic genetic information of Korean pigs. The swine leukocyte antigen - 2 (SLA-2) gene in the MHC classical region was cloned with spleen tissues from Korean native pigs selected from the main land (KNP) and Jeju Island (KJP). Primer sequences based on swine cDNA (GenBank accession numbers AF464049 and AF464005) were used to amplify the entire SLA-2 gene, and the amplification product including both 3′ and 5′ UTRs was sized 1,520 bp. A BAC clone was selected from miniature pigs and sequenced for the genomic region of the SLA-2 gene showing that 4,585 bp in total length consisted of exons (1,087 bp) and introns (3,498 bp). A sequence analysis confirmed 58 SNPs in coding regions, which revealed higher numbers of SNPs in KNP than other pig breeds, implicating more genetic variability in Korean pigs. Approximately 82% of the SLA-2 SNPs were located in the highly polymorphic exons 2 and 3. Newly identified sequences of the SLA-2 gene for KNP and KJP were submitted into the IPD-MHC database with new nomenclatures (SLA-2*1501, SLA-2*1601, and SLA-2*w08hy01 allele), while the representative sequences of KNP and KJP were submitted into GenBank with accession numbers (DQ992495, DQ992496, and DQ992501), respectively. The identified KNP allele (SLA-2*1501) clustered with previously defined alleles for Korean pigs (SLA-2*kn02 and SLA-2*jh01), but SLA-2*1601 (KNP) and SLA-2*w08hy01 (KJP) alleles showed no significant genetic relationships with any other allele. A sequence comparison revealed that KNP has departed from KJP both genetically and phenotypically. The results of SLA-2 SNP in KNP and KJP reported here will serve as the SLA-2 reference for Korean pigs.     

Cytogenetic analysis of the X chromosome region 2B3-4 — 2B11 of Drosophila melanogaster

Mutation t467, belonging to the swi complementation group, and causing death in late prepupa, is located in the interval from 2B6 to the left part of 2B7-8. In this region puffing is absent in salivary gland chromosomes. In t467/t467 homozygotes intermoult early and early-late larval 20-OH ecdysone puffs do not differ from the controls. Mid-prepupal puffs are normal too with a few exceptions. However, all late larval and prepupal puffs are reduced or absent in the mutant. Both, hormone incubation of t467 glands in vitro and hormone injection have shown: i) 20-OH ecdysone in vitro does not restore the normal larval puffing pattern. ii) Withdrawal of the hormone from glands at PS6 causes premature appearance of late larval puffs, which, however, do not reach control sizes. It is concluded that the swi gene product is necessary for induction of late puffs. Thus in the 2B3-4—2B7-8 region three genes, affecting 20-OH ecdysone induction processes, have become known.     

What are the implications of evolvable molecules?

James Shapiro’s view of evolution is inspired by looking at the molecular mechanisms of mutation. Finding these systems to be intelligent and the mutations non-gradual, Shapiro concludes that neither the role of DNA in development, nor and the role of natural selection in evolution are what we thought them to be. The cases discussed are interesting and may require some modification of theory in biology, but this reviewer finds many of Shapiro’s conclusions unwarranted.     

Residue 3 of β2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody

 Previous studies of class I MHC molecules have shown that the owl monkey (Aotus) possesses at least two variants of the β₂-microglobulin (β₂m) protein. These two variants have different isoelectric points, and exhibit differential reactivity with the monoclonal antibody W6/32. We report cDNA sequences of the B2m gene, from W6/32-positive and W6/32-negative Aotus cell lines. The two β₂m variants we identified exhibit a single amino acid difference at position three. An arginine residue at position 3 was correlated with W6/32 reactivity, whereas histidine was associated with non-reactivity. W6/32 reactivity was conferred to a W6/32-negative Aotus cell line when it was transfected with the B2m from the W6/32-positive cell line. Residue 3 of β₂m is located at the surface of the class I molecule. It is also close to position 121 of the MHC class I heavy chain, which has previously been shown to influence W6/32 antibody binding. We conclude that W6/32 binds a compact epitope on the class I molecule that includes both residue 3 of β₂m and residue 121 of the heavy chain. We examined the distribution of the two β₂m motifs in a sample Aotus population using an allele-specific polymerase chain reaction assay. The pattern of β₂m segregation we observed matches that which was defined previously by serology. Additionally, we identified laboratory-born hybrid animals who possess both variants of β₂m. Received: 1 April 1998 / Received: 3 July 1998     

Novel non-ATP competitive small molecules targeting the CK2 α/β interface

Increased CK2 levels are prevalent in many cancers. Combined with the critical role CK2 plays in many cell-signaling pathways, this makes it a prime target for down regulation to fight tumour growth. Herein, we report a fragment-based approach to inhibiting the interaction between CK2α and CK2β at the α-β interface of the holoenzyme. A fragment, CAM187, with an IC₅₀ of 44?μM and a molecular weight of only 257?gmol^?1 has been identified as the most promising compound. Importantly, the lead fragment only bound at the interface and was not observed in the ATP binding site of the protein when co-crystallised with CK2α. The fragment-like molecules discovered in this study represent unique scaffolds to CK2 inhibition and leave room for further optimisation.     

Kinetics of paclitaxel 2′-N-methylpyridinium mesylate decomposition

This study was designed to examine the kinetics of decomposition of paclitaxel 2′-N-methylpyridinium mesylate (PNMM), a derivative of paclitaxel. Further, the potential for PNMM to act as a prodrug of paclitaxel was assessed in vitro. Stability studies of PNMM were conducted over a pH range of 4.0 to 8.0 at 25°C. The critical micelle concentration (CMC) of PNMM was determined by pulsating bubble surfactometry. Studies of the conversion of PNMM to paclitaxel were conducted in vitro in human plasma. Decomposition of PNMM followed apparent zero-order kinetics. The pH-rate profile exhibited no evidence of acid catalysis down to pH 4.0, while the rate was accelerated under base conditions. Surface tension studies suggested that PNMM formed micelles with a CMC of approximately 34 μg/mL. Conversion studies in phosphate buffer showed that no more than 5% of PNMM converted to paclitaxel, while in human plasma the conversion was about 25%. The degradation of PNMM was via apparent zero-order kinetics and was dependent upon pH. The observed apparent zero-order kinetics of decomposition of PNMM was consistent with the formation of micelles in phosphate buffer. In buffered aqueous media alone or in human plasma, PNMM did not convert quantitatively to paclitaxel. Thus, the limiting factor in the application of PNMM as a prodrug would appear to be the poor potential to convert to paclitaxel. This article was presented in part at the AAPS Annual Meeting and Exposition, New Orleans, LA, November 14–18, 1999.     

Comprehensive analysis of the roles of ‘black’ and ‘gray’ clusters in structure and function of rat β-parvalbumin

Recently we found two highly conserved structural motifs in the proteins of the EF-hand calcium binding protein family. These motifs provide a supporting scaffold for the Ca²⁺ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif forms a cluster of three amino acids called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster). Cluster I is much more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II includes a mix of aromatic, hydrophobic, and polar amino acids. The ‘black’ and ‘gray’ clusters in rat β-parvalbumin consist of F48, A100, F103 and G61, L64, M87, respectively. In the present work, we sequentially substituted these amino acids residues by Ala, except Ala100, which was substituted by Val. Physical properties of the mutants were studied by circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probe methods. The Ca²⁺ and Mg²⁺ binding affinities of these mutants were evaluated by intrinsic fluorescence and equilibrium dialysis methods. In spite of a rather complicated pattern of contributions of separate amino acid residues of the ‘black’ and ‘gray’ clusters into maintenance of rat β-parvalbumin structural and functional status, the alanine substitutions in the cluster I cause noticeably more pronounced changes in various structural parameters of proteins, such as hydrodynamic radius of apo-form, thermal stability of Ca²⁺/Mg²⁺-loaded forms, and total energy of Ca²⁺ binding in comparison with the changes caused by amino acid substitutions in the cluster II. These findings were further supported by the outputs of computational analysis of the effects of these mutations on the intrinsic disorder predisposition of rat β-parvalbumin, which also indicated that local intrinsic disorder propensities and the overall levels of predicted disorder were strongly affected by mutations in the cluster I, whereas mutations in cluster II had less pronounced effects. These results demonstrate that amino acids of the cluster I provide more essential contribution to the maintenance of structuraland functional properties of the protein in comparison with the residues of the cluster II.     

Response analysis of the lumbar spine during regular daily activities—A finite element analysis

A non-linear poroelastic finite element model of the lumbar spine was developed to investigate spinal response during daily dynamic physiological activities. Swelling was simulated by imposing a boundary pore pressure of 0.25 MPa at all external surfaces. Partial saturation of the disc was introduced to circumvent the negative pressures otherwise computed upon unloading. The loading conditions represented a pre-conditioning full day followed by another day of loading: 8 h rest under a constant compressive load of 350 N, followed by 16 h loading phase under constant or cyclic compressive load varying in between 1000 and 1600 N. In addition, the effect of one or two short resting periods in the latter loading phase was studied.The model yielded fairly good agreement with in-vivo and in-vitro measurements. Taking the partial saturation of the disc into account, no negative pore pressures were generated during unloading and recovery phase. Recovery phase was faster than the loading period with equilibrium reached in only ～3 h. With time and during the day, the axial displacement, fluid loss, axial stress and disc radial strain increased whereas the pore pressure and disc collagen fiber strains decreased. The fluid pressurization and collagen fiber stiffening were noticeable early in the morning, which gave way to greater compression stresses and radial strains in the annulus bulk as time went by. The rest periods dampened foregoing differences between the early morning and late in the afternoon periods. The forgoing diurnal variations have profound effects on lumbar spine biomechanics and risk of injury.