Biological Events in Periodontal Ligament and Alveolar Bone Associated with Application of Orthodontic Forces.

Orthodontic force-induced stresses cause dynamic alterations within the extracellular matrix and within the cytoskeleton of cells in the periodontal ligament and alveolar bone, mediating bone remodelling, ultimately enabling orthodontic tooth movement. In the periodontal ligament and alveolar bone, the mechanically induced tensile strains upregulate the expression of osteogenic genes resulting in bone formation, while mechanically induced compressive strains mediate predominantly catabolic tissue changes and bone resorption. In this review article we summarize some of the currently known biological events occurring in the periodontal ligament and in the alveolar bone in response to application of orthodontic forces and how these facilitate tooth movement.

Periodontal Biological Events Associated with Orthodontic Tooth Movement: The Biomechanics of the Cytoskeleton and the Extracellular Matrix.

The mechanical stimuli generated by orthodontic forces cause deformation of extracellular matrices and cells, vascular changes, inflammation, and the release of active biological agents generating a complex multifactorial sequence of biological events culminating in bone remodelling enabling orthodontic tooth movement. Orthodontic forces on the teeth generate stresses in periodontal tissues according to a number of variables including the type (continuous, interrupted, or intermittent), magnitude, direction, and frequency of the applied load. Whether the strain is compressive or tensile determines whether bone deposition or bone resorption will occur. The mechanically induced strains mediate structural changes in extracellular matrices and in cells, consequently affecting cellular gene expression and function. In the extracellular matrix, mechanosensing molecules integrated into the structure of various proteins can be activated upon load-induced protein unfolding. These specialized molecules have the capacity to sense and then to convert microenvironmental biomechanical stimuli into intracellular biochemical signals that interact to generate a coordinated tissue response. It is also possible that the applied force may directly cause nuclear deformation with configurational changes in chromatin, thus influencing gene expression. In this review article we summarize the current general concepts of mechanotransduction influencing the remodelling of periodontal tissues thus enabling tooth movement in response to applied orthodontic loads.

Osseointegration: biological events in relation to characteristics of the implant surface.

Osseointegration of titanium implants is a complex biological process involving interactions between immuno-inflammatory responses, angiogenesis and osteogenesis, all of which are influenced by the physical and chemical characteristics of the implant surface. An implant surface with moderately rough topography and high surface energy influences cellular activities, enhancing peri-implant bone wound healing. Primary mechanical stability of the implant is essential for osseointegration. In this article we review some of the more important biological events of peri-implant bone wound healing in the process of osseointegration, and discuss how the biophysical properties of implant surfaces influence cellular responses.

Antigenic competition between polypeptidyl determinants in normal and tolerant rabbits.

Competition between two polypeptidyl determinants was studied in normal rabbits and rabbits made tolerant to the competing antigen. The capacity of poly-DL-phenylalanyl protein conjugate to inhibit the formation of antibodies specific to the poly-DL-alanyl determinant was dependent on the nature of the protein carrier of the singly substituted antigens. Competition occurred only when the peptidyl determinants were attached to identical or similar (RSA and HSA) carriers. Thus, the immune response toward the poly-DL-alanyl determinant was impaired by injecting the pairs p-DL-PheRSA and p-DL-AlaHSA, or p-DL-PheRNase and p-DL-AlaRNase. Suppression of the formation of antibodies with poly-DL-alanyl specificity was not observed, however, upon administration of p-DL-PheRSA together with p-DL-AlaRNase or of p-DL-PheRNase with p-DL-AlaHSA. Tolerance to p-DL-PheRSA was induced by injecting this material into newborn rabbits. The tolerant animals retained their capacity to produce anti-poly-DL-alanyl antibodies upon injection of p-DL-AlaRSA or p-DL-AlaHSA. However, when these poly-DL-alanyl proteins were administered together with p-DL-PheRSA, antibodies against the poly-DL-alanyl determinant were not formed even though no antibodies with poly-DL-phenylalanyl specificity were produced. These results indicate that in competition experiments the preference in the immune response against a given determinant is dependent not only on the nature of the competing determinants, but it is also governed to a large extent by the over-all properties of the antigenic molecules. This suggests that at the stage at which the competition occurs the competing molecules had not undergone considerable degradation. On the basis of experiments with tolerant animals, it is suggested that in normal animals antibody formation to the competing antigen is not the cause of its inhibitory action on the response against the other antigen. The competition experiments described suggest that an antibody-forming cell is multipotent.

Partial amino acid sequence of the precursor of immunoglobulin light chain programmed by messenger RNA in vitro.

The five proteins programmed in a cell-free system by a mouse kappa light chain messenger RNA were labeled with [3H]leucine and subjected to amino acid sequence analyses. In all five proteins, 20 amino acid residues precede the amino terminus of the mature protein, indicating that there is one major point for the initiation of messenger RNA translation. The abundance (30 percent) of leucine residues in the extra piece (leucine at positions 6, 7, 8, 11, 12, and 13) indicates that this moiety is hydrophobic. Furthermore, it seems that the precursor may have an additional extra piece at the carboxyl terminus.

Activity of the promoter of the hsp70 gene of the parasitic helminth Schistosoma mansoni in the mammalian CHO cell-line.

Functional assay of schistosome promoters is problematic because parasite cell-lines are not available. We found that the hsp70 promoters of S. mansoni and of other eukaryotes reveal similarities in the sequence and organization of the regulatory elements. Therefore, a construct of the schistosome hsp70 promoter linked to the CAT reporter gene was prepared and used to transfect CHO cells. The transfected cells showed heat shock dependent activation of CAT. These findings indicate that functional evaluation of schistosome promoters can be done, in part, in foster cell-lines.

Expression of different forms of the heat-shock factor during the life cycle of the parasitic helminth Schistosoma mansoni.

HSE-HSF complexes from different stages of the life cycle of schistosome were analyzed by pore exclusion electrophoresis (estimates the size of the native DNA-protein complex), and by U.V.-cross-linking followed by SDS-PAGE (estimates the size of the monomeric heat shock factor, HSF). The apparent M(r) values of the native and monomeric HSE-HSF complexes from schistosomula were 80,000 and 60,000, respectively. In adult worms M(r) values were 70,000 for the native HSE-HSF complex, 60,000 and 80,000 for the monomeric form. These findings indicate that: (1) schistosome express two (maybe three) forms of HSF, (2) different forms of HSF are expressed at different developmental stages. The findings that the native HSE-HSF complex and the monomeric HSF are of a similar size indicates that the complex contains a single HSF, or that the complex is a labile oligomer of HSF that decomposes into monomers during electrophoresis in a nondenaturing gel.

Differences in DNA sequence recognition by the heat-shock factors of Drosophila melanogaster and the parasitic helminth Schistosoma mansoni.

It was recently shown that schistosome extracts contain heat-shock factor (HSF) activity that correlates with the pattern of hsp70 mRNA levels at different developmental stages of the parasite (Levy-Holtzman and Schechter (1994) Parasitology 108, 35-42). To extend our understanding of the HSF activity revealed in extracts of Schistosoma mansoni (Sm), it was further analyzed by competition experiments and compared with the well characterized HSF of Drosophila melanogaster (Dm). The interactions of HSF in Sm extracts (SmHSF) and HSF of Dm (DmHSF) with 32P-labeled heat shock element (HSE) probes, with and without unlabeled competitor DNA probes (HSE-related oligos), were analyzed by gel retardation assay. The binding and inhibition studies demonstrated that SmHSF and DmHSF differ in HSE sequence recognition: an array of three nGAAn inverted repeats according to the ideal consensus sequence (nGAAnnTTCnnGAAn) is recognized by DmHSF, but not by SmHSF. In the schistosome, binding is attained only when the third pentamer is a variant, composed of nGTAn instead of nGAAn. The presence of this variant in the promoter of the hsp70 gene of the parasite suggests coevolution of the variant sequence together with the SmHSF which interacts efficiently with the variant, but not with the ideal HSE sequence. Further inhibition studies revealed additional differences between SmHSF and DmHSF in recognition of the first and second nGAAn pentamers of HSE. In analogy to other systems of ligand-protein interactions, we propose that the complementarity between the HSE ligand and the HSF protein is higher in SmHSF, as compared to DmHSF.

Cloning and characterization of the SmIMP25 integral membrane protein of the parasitic helminth Schistosoma mansoni.

The cDNA and genomic clones encoding a 25 kDa integral membrane protein, termed SmIMP25, were isolated from Schistosoma mansoni. The 2.2 kb SmIMP25 mRNA was found in all developmental stages of the parasite tested: miracidium, sporocyst, cercaria and adult worm. The SmIMP25 gene is at least 16 kb long and it is split by four introns ranging in size from 36 bp to > or = 9 kb. Excluding the introns, the gene and the cDNA show 100% sequence identity. The cDNA has an open reading frame encoding a protein 223 amino acids long. The predicted sequence reveals a distinct hydrophobic domain of 20 amino acids located 12 residues from the carboxyl-terminal end. The properties of this domain (marked hydrophobicity, size, flanking by charged residues and C-terminal location) are typical of the transmembrane segments of integral membrane proteins. The presence of three potential N-glycosylation sites is also consistent with membrane proteins that are often glycosylated at the extracellular domain. Accordingly we propose that SmIMP25 is an integral membrane protein in which residues 1-191 are extracellular, residues 192-211 comprise the hydrophobic domain that spans the membrane, and residues 212-223 are intracellular. The SmIMP25 was synthesized as a fusion protein in bacteria and antibodies were elicited in rabbits. Antibodies against SmIMP25 specifically precipitated a 25 kDa protein from cell-free products programmed by schistosome mRNA, in agreement with the size of the protein predicted from the cDNA sequence. Immunofluorescence studies showed SmIMP25 on the surface of the parasite. Surface molecules expressed at the host-parasite interface are likely to provide information on host parasite relationship and may serve as targets for protective immunity.

Cloning of the SmSPO-1 gene preferentially expressed in sporocyst during the life cycle of the parasitic helminth Schistosoma mansoni1.

Schistosomes are parasitic helminths with a complex life cycle in human and snail hosts. They express stage-specific genes that conceivably determine distinct properties of the parasite at different developmental stages. Here we report the stage-specific gene SmSPO-1, which is preferentially expressed in sporocysts residing in the snail host. The cDNA and the gene were cloned and sequenced. The cDNA, from cap site to the poly(A) addition site, is 498 bp long. It encodes a protein of 117 amino acids with a hydrophobic signal peptide of 18 residues, indicating that SmSPO-1 is a secreted or a membranal protein. In the gene the cDNA is split into four exons spread over 2.1 kb of chromosomal DNA.

Differences in DNA-sequence recognition between the DNA-binding domain fragment and the full-length molecule of the heat-shock transcription factor of schistosome.

Binding and inhibition studies reveal that the DNA-binding domain (DBD) fragment and the full-length molecule of the heat-shock transcription factor of schistosome (SmHSF) differ in DNA sequence recognition. SmHSF does not recognize the ideal HSE consensus sequence (nGAAnnTTCnnGAAn) but recognizes a variant HSE that contains nGTAn instead of nGAAn in the third pentamer. The DBD reacts efficiently with the ideal HSE sequence and with lower affinity with the variant HSE sequence. These findings suggest that elements inside and outside the DBD contribute to the DNA-binding specificity of HSF.

Mapping of the active site of proteases in the 1960s and rational design of inhibitors/drugs in the 1990s.

For several decades the specificity of proteases has been presented as an active site divided into subsites, using the nomenclature of Schechter & Berger from 1967 (S1, S2... for subsites of the active site; P1, P2... for residues of the substrate occupying the corresponding subsites). At early stages of the research (1960s) it was realized that the size of the active site was larger than expected and important interactions occur in regions remote from the catalytic site. Since the active site was found to be large it was divided into subsites, and a procedure to map it up was developed. The map provides information on the size of the active site (number of subsites), the properties of each subsite (free energy of ligand binding, nature of binding forces, etc.), and it enables rational design of new substrates and inhibitors. Already in 1968 inhibitors with binding constants ten thousand fold higher than available inhibitors, were prepared. The model of a large active site was initially met with strong opposition. Before long, however, predictions of the model (size of the active site, interactions in subsites remote from the catalytic site) were confirmed by X-ray crystallography (1970). During the 1990s proteolytic enzymes received renewed attention in biology and medicine, they became therapeutic targets, and protease inhibitors were successfully applied in the treatment of AIDS and hypertension. The model of large active site divided into subsites, proposed 38 years ago, stood the test of time. This model is still in use in basic research to evaluate enzyme activity, and in pharmaceutical research for the development of inhibitors/drugs.

Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse.

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.

Dysfunction of early-stage visual processing in schizophrenia.

OBJECTIVE: Schizophrenia is associated with deficits in higher-order processing of visual information. This study evaluated the integrity of early visual processing in order to evaluate the overall pattern of visual dysfunction in schizophrenia. METHOD: Steady-state visual-evoked potential responses were recorded over the occipital cortex in patients with schizophrenia and in age- and sex-matched comparison volunteers. Visual-evoked potentials were obtained for stimuli composed of isolated squares that were modulated sinusoidally in luminance contrast, number of squares, or chromatic contrast in order to emphasize magnocellular or parvocellular visual pathway activity. RESULTS: Responses of patients to magnocellular-biased stimuli were significantly lower than those of comparison volunteers. These lower response levels were observed in conditions using both low luminance contrast and large squares that biased processing toward the magnocellular pathway. In contrast, responses to stimuli that biased processing toward the parvocellular pathway were not significantly different between schizophrenia patients and comparison volunteers. A significant interaction of group and stimulus type was observed in the condition using low luminance contrast. CONCLUSIONS: These findings suggest a dysfunction of lower-level visual pathways, which was more prominent for magnocellular than parvocellular biased stimuli. The magnocellular pathway helps in orienting toward salient stimuli. A magnocellular pathway deficit could contribute to higher-level visual cognitive deficits in schizophrenia.

Converting spatial to pseudotemporal resolution in laser plasma analysis by simultaneous multifiber spectroscopy

Traditional chemical analysis based on laser plasma spectroscopy (LPS) requires time-gated detectors, to avoid the initial signal from the hot plasma. These detectors are expensive and often need to be cooled and protected against vapor condensation. We suggest a low-cost setup that may replace these gated detectors, while maintaining acceptable analytical performance. The proposed setup is a result of investigation of plasma-front propagation in LPS analysis. It is known that the LPS plasma propagation is similar to the shock wave propagation after a strong explosion in the atmosphere. We found that the propagation of the plasma fits well the Sedov blast wave theory, providing a good agreement between the theoretical and experimental figures. A proper observation geometry, which is perpendicular to the plasma expansion vector, enables converting spatial to temporal resolution. We take advantage of the fact that the plasma reaches a given distance above the analyzed surface at a certain time delay. Therefore, a single optical fiber, positioned at a well-defined geometry, can provide spectral information corresponding to a certain time delay. A multifiber imaging spectrometer provides information corresponding to a series of delay times, which is adequate for analysis of a variety of matrixes. It was found that the performance of the nongated detector observing a narrow solid angle is similar to that of a gated one observing the whole plasma. For one particular example, observing the plasma from a distance of 4.5 mm is equivalent to a delay of 4 micros and integration time of 2 mircos. The ratio of spectral lines of two elements was investigated using the spatially resolved (nongated) setup, and it was found that this mode is advantageous when internal calibration is applied. It was concluded that sensitive LPS analyses can be carried out by less expensive (nongated) detectors.

Cloning and sequencing of an hsp70 gene of Schistosoma mansoni.

Schistosomes have a complex life cycle (vertebrate and molluscan hosts as well as larvae living freely in water) in which they are exposed to different environments and temperatures (20 degrees C - 37 degrees C). Since heat shock genes are activated in response to stress and during development [1], it is of interest to study the hsp70 gene family in schistosome. To approach this issue we have isolated from Schistosoma mansoni a genomic clone containing the complete coding region of hsp70 and the 5' flanking DNA with transcription regulatory elements including HSE (heat shock element) sequences.

Rapid changes in the expression of a gene encoding a calcium-binding protein in Schistosoma mansoni.

Genes expressed in a stage-specific manner may help us understand the molecular events controlling the complex life cycle of schistosomes. cDNA and genomic clones encoding a calcium-binding protein (CaBP) were obtained from cercariae and their sequence determined. The encoded protein (69 amino acids long) shows clear resemblance to the domain structure and organization of CaBP molecules. It contains two typical calcium-binding loops, the distance between which is identical to the length conserved in other CaBP molecules. In addition, the schistosome CaBP shows Ca2+-dependent electrophoretic mobility (increased with Ca2+-ions and decreased with EGTA). Northern blots revealed expression of the CaBP gene in cercariae but not in sporocyst or worm (developmental stages preceding and following cercaria). The preferential expression of this CaBP in the cercaria raises questions as to what cercaria-specific function(s) it performs. The structure of the gene is similar to that in other eukaryotes, and one intron interrupts the coding sequence. The region of the cap site was determined, and there was no evidence of the spliced leader sequence found in the mRNAs of other parasites. The CaBP reveals a rapid change in gene expression, since the mRNA is missing in the parasite residing in infected snails, but is readily detected in cercariae 1 h after shedding. We identified other genes which are turned on (like the CaBP) or shut off within the short period of transition from cercariae in the snail to free-swimming cercariae.

Structure of the gene encoding a putative Schistosoma mansoni tegumental antigen precursor.

In order to obtain the complete gene encoding the putative precursor of a 15-kDa Schistosoma mansoni tegumental antigen (Sm15), two cDNAs (A70 and A184) and two fragments of independent genomic clones were subcloned and sequenced. The collated sequence contains 4700 nucleotides and represents the full length open reading frame of the gene, encoding a protein of 1032 amino acids with a calculated molecular mass of 116,900. Thus, the gene encodes a much longer protein than that identified in the tegumental membranes, suggesting that it encodes a precursor that is subsequently highly processed. A 964-bp region composed of 5 closely related repeats was found to be present within the translated frame. The predicted protein is highly acidic and there is no indication of hydrophobic domains that may represent transmembrane regions or indicate attachment of a GPI anchor. The coding region has no homologies in the currently available data bases. In the 5' non-transcribed area a copy of the SM alpha repeat family is present. The coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression.

Photochemical study of anthracene crystallites by Fourier transform spectroscopic imaging.

Fourier transform-based spectroscopic imaging was used for direct, time-resolved, analysis of UV-irradiated anthracene crystallites. Well-resolved fluorescence spectra were obtained at a spatial resolution of 1 microm. The appearance of such photochemical by-products as dianthracene and anthraquinone was monitored throughout the irradiation experiments. Under deaerated conditions, photolysis of anthracene was accompanied by formation of dianthracene. When performed under aerated conditions, however, the spectral data indicated formation of both dianthracene and anthraquinone. Spectral features obtained for the directly monitored photolysis of anthracene are discussed in respect to the structural and compositional modifications in such crystallites. Capabilities of the spectral imaging device for the quantification of the photochemical products of anthracene are discussed.

The use of biological dressings in radical vulvectomy.

The use of biological dressing techniques accelerates recovery from radical vulvectomy combined with lymphadenectomy. Our earliest interest in the field arose as a result of the morbidity rate which ensued subsequent to surgical therapy of cancer of the vulva. The technique involved in 8 such procedures is described, and its applicability illustrated with photographic plates and review of the literature.