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Biomedical subjects

S Winkler

Publications and source records attributed to S Winkler.

At least 73 records · Page 4Linked to original sources
PubMed2000

Reduction-oxidation control of beta-sheet assembly in genetically engineered silk.

Genetically engineered spider dragline silk protein was modified to incorporate methionines flanking the beta-sheet forming polyalanine regions. The methionines could be selectively chemically oxidized and reduced. This chemical change altered the bulkiness and charge of the sulfhydryl groups, and in turn, the beta-sheet forming tendencies of the polyalanine domains and solubility of the protein. The genes encoding these redesigned proteins were constructed, cloned and expressed in Escherichia coli. In the reduced state (beta-mercaptoethanol) the approximately 25 kDa protein behaved similarly to native spider dragline silk, crystallizing into beta-sheets based on diffraction analysis and appearing fibrous by TEM. The addition of the methionines into the consensus dragline silk sequence did not disrupt the normal macromolecular assembly behavior of the protein. In the oxidized state (phenacyl bromide) the protein did not form beta-sheet crystals and appeared morphologically featureless based on TEM. A reduction in beta-strand content was also observed upon oxidation based on FTIR and TEM analysis and confirmed by X-ray diffraction analysis. To further confirm changes in assembly behavior observed for the recombinant protein containing the methionines, a model peptide with the same repeat amino acid sequence was synthesized and characterized. Shifts in molecular weight, observed by MALDI, along with corresponding changes in crystallinity, by electron diffraction, agreed with the changes expected on activation and deactivation of the redox trigger. These results support the use of a redox trigger as a useful feature with which to control the assembly of beta-sheet forming proteins.

Amino Acid Sequence↗
PubMed2000

CD 95-independent mechanisms of IL-2 deprivation-induced apoptosis in activated human lymphocytes.

Growth factor deprivation-induced apoptosis plays an important role in several cellular systems. However, knowledge of the molecular mechanisms involved are restricted to a few murine models or tumor cell lines. Therefore, we aimed studying signaling pathways leading to apoptosis in activated human peripheral T cells after IL-2 withdrawal. Lymphoblasts from patients with CD 95 (Fas/APO-1)-deficiency revealed that functional CD95 was not required to induce apoptosis after IL-2 withdrawal. Moreover, apoptosis induction in response to various cytotoxic stimuli was found to be mediated in the absence of functional CD95 but was affirmatorily influenced by IL-2 signaling. Immunoblots showed no downregulation of Bcl-2 or Bcl-xL and no upregulation of Bax, whereas decreased mitochondrial membrane potential was readily measurable 24 h after cytokine deprivation. Tetrapeptide inhibitors showed limited efficacy in preventing apoptosis whereas the caspase inhibitor zVAD-FMK potently blocked induction of apoptosis. Cleavage of different fluorogenic substrates revealed multiple caspase enzyme activities in lymphoblasts, which were not negatively affected by the fas mutation. Starting at 8 h after IL-2 withdrawal, upregulation of active caspase-3 but not of caspase-8 could be detected. Taken together, our data argue for molecular mechanisms of cytokine deprivation-induced apoptosis in activated human lymphocytes independent of CD95.

Apoptosis↗
PubMed2000

The conditional medaka mutation eyeless uncouples patterning and morphogenesis of the eye.

In early vertebrate eye development, the retinal anlage is specified in the anterior neuroectoderm. During neurulation, the optic vesicles evaginate from the lateral wall of the prosencephalon. Here we describe the temperature-sensitive mutation eyeless in the Japanese medakafish. Marker gene analysis indicates that, whereas, specification of two retinal primordia and proximodistal patterning takes place in the mutant embryo, optic vesicle evagination does not occur and subsequent differentiation of the retinal primordia is not observed. The mutation eyeless thus uncouples patterning and morphogenesis at early steps of retinal development. Temperature-shift experiments indicate a requirement for eyeless activity prior to optic vesicle evagination. Cell transplantation shows that eyeless acts cell autonomously.

Animals↗
PubMed2000

The ankylos endosseous dental implant: assessment of stability up to 18 months with the Periotest.

Osseointegration is an ongoing histometric process that may vary during clinical function. The implant must be stable at uncovering, which reflects the status of bone-implant interface. The physiology of bone healing associated with endosseous implants suggests that this process occurs between 8 and 12 months, and Periotest values (PTVs) tend to reflect changes in the stability of the bone-implant interface. Stability generally increases gradually from the time of uncovering to an optimal PTV that occurs at a point close to 12 months. This stable interface must remain intact for long-term clinical survival. Rapid development of this optimal PTV is highly desirable in order to prevent premature overloading of the bone-implant interface. The Ankylos implant is a new screw-type implant design in which the thread pitch and length vary to maximize trabecular bone contact. The purpose of this report is to evaluate to 18 months the stability (PTVs) of this implant design. More than 457 implants were placed and followed for a period of 18 months by the multicentered, multidisciplinary Ankylos Implant Clinical Research Group (AICRG). Implant stability (PTVs) was assessed using the Periotest at abutment connection and at 3, 6, 9, 12, and 18 months after uncovering. The Periotest values for all implants rapidly reached an optimal status between uncovering (-3.1 PTVs) and 3 months (-3.4 PTVs). This rapid increase in stability has not previously been reported for other implant designs. The mandibular arch was more negative (-3.8 PTVs) at uncovering as compared with the PTVs for the maxillary arch (-1.7 PTVs). Negative PTVs were recorded (1) as length and diameter increased, (2) as bone density increased, (3) in certain jaw regions, (4) as the number of implants/case increased, and (5) for implants stabile at placement. The Ankylos screw implant design produced rapid stabilization 3 months after uncovering.

Analysis of Variance↗
PubMed2000

Implant survival in patients with type 2 diabetes: placement to 36 months.

BACKGROUND: Because the life expectancy of individuals continues to increase, dentists providing dental implant treatment can expect to see an increasing number of patients with diabetes mellitus. Today, there are little data available concerning the clinical outcomes involving the use of implant treatment for patients with diabetes mellitus. There are three types of diabetes mellitus: Type 1 (insulin dependent), Type 2 (non-insulin dependent), and gestational. Because of possible complications from patients with diabetes mellitus, they are excluded from participation in most clinical studies of endosseous dental implant survival. METHODS: This study attempted to determine if Type 2 diabetes represents a significant risk factor to the long-term clinical performance of dental implants, using the comprehensive DICRG database. Diabetes was a possible exclusion criterion; however, the final decision on Type 2 patients was left to the dental implant team at the research center. A total of 2,887 implants (663 patients) were surgically placed, restored, and followed for a period of 36 months. Of these, 2,632 (91%) implants were placed in non-diabetic patients and 255 (8.8%) in Type 2 patients. Failures (survival) were compared using descriptive data. Possible clustering was also studied. RESULTS: A model assuming independence showed that implants in Type 2 patients have significantly more failures (P = 0.020). However, if correlations among implants within the patient are considered, the significance level becomes marginal (P = 0.046). The experience of the surgeon did not produce a clinically significant improvement in implant survival. The use of chlorhexidine rinses following implant placement resulted in a slight improvement (2.5%) in survival in non-Type 2 patients and a greater improvement in Type 2 patients (9.1%); the use of preoperative antibiotics improved survival by 4.5% in non-Type 2 patients and 10.5% in Type 2 patients. The use of HA-coated implants improved survival by 13.2% in Type 2 diabetics. CONCLUSION: Type 2 diabetic patients tend to have more failures than non-diabetic patients; however, the influence was marginally significant. These findings need to be confirmed by other scientific clinical studies with a larger Type 2 diabetic sample size.

Adult↗
PubMed2000

Implant survival to 36 months as related to length and diameter.

BACKGROUND: It is generally accepted that diameter and length of an endosseous dental implant and its stability at placement are critical factors in achieving and maintaining osseointegration. In the event of slight implant mobility at placement, the conventional or accepted treatment is to place a longer implant and/or one of wider diameter. This manuscript presents stability and survival/failure data for implants of different diameters and lengths following 36 months post-placement, as well as crestal bone loss data between placement and uncovering. METHODS: A subset of the Dental Implant Clinical Research Group's database was used to study the 3-year survival and stability of various implant lengths (7 mm, 8 mm, 10 mm, 13 mm, and 16 mm) and diameters (3 mm+ and 4 mm+). Placement to uncovering crestal bone loss was also determined. The implants were generally representative of those available for clinical use (screws, basket, grooved, hydroxy-apatite-coated, CP-Ti, Ti-alloy). The study protocol specified that the implants be randomized to various jaw regions to accomplish the primary goals of the study--the comparison of each implant design's overall survival. A total of 2,917 implants were placed, restored, and followed. Data for all 3 mm to 3.9 mm diameter implants were pooled into a "3+" group, and the 4 mm to 4.9 mm diameter implants into a "4+" mm group. No attempt was made to look at the influence of any other variables on survival outcomes. The possible influence of clustering on survival was taken into consideration. RESULTS: The 3+ mm group had a mean stability (PTV) of -3.8 (SD = 2.9), and the 4+ group had a mean PTV of -4.4 (SD = 2.7) (P < 0.05). The PTVs for implant lengths ranged from -2.9 (SD = 2.8) for 7 mm lengths to -3.9 (SD = 2.9) for 16 mm lengths (P < 0.05). Survival to 36 months was 90.7% for the 3+ diameter and 94.6% for the 4+ group (P = 0.01). Survival ranged from 66.7% for the 7 mm implants to 96.4% for 16 mm implants (P = 0.001). Outcomes did not change when clustering was considered, although the P value decreased slightly. CONCLUSIONS: The results indicate that: 1) shorter implants had statistically lower survival rates as compared with longer implants; 2) 3+ mm diameter implants had a lower survival rate as compared with 4+ mm implants; 3) 3+ mm diameter implants are less stable (more positive PTVs) than 4+ mm implants; and 4) there was no significant difference in crestal bone loss for the two different implant diameters between placement and uncovering.

Alloys↗
PubMed2000

Congenital macroglossal angiodysplasia ("Lymphangioendotheliomatosis").

A case of congenital lingual angiodysplasia with macroglossia in a 5-year-old girl is presented. A diffusely enlarged tongue was present at birth and continued to grow as the child aged. It was accompanied by impaired speech, difficulty in eating and breathing, and sleep apnea, necessitating surgical intervention. The fundamental lesion represents a complex vascular malformation of the lymphangioma-hemangioma type, involving extensively the deep musculature of the tongue. Multifocal and multicentric cavernous lymphangioma-like and hemangioma-like areas merge with benign angioendotheliomatous-like foci in a background of variable muscle degeneration and marked fibrosis. Neither a borderline nor an overtly malignant vasoformative neoplasm was present. Because of its distinctively widespread, multicentric intramuscular distribution, this lesion may be construed as a diffuse variant of lingual lymphangioma-hemangioma malformation, closely resembling a previously described case of macroglossal lymphangioendotheliomatosis.

Arteriovenous Malformations↗
PubMed2000

[Reciprocal modification of Fas activation and stress protein response decides apoptosis or resistance development of cells].

Activation of heat shock factor (HSF)-1 DNA binding and heat shock protein (hsp)-70 expression enable resistance of cells to various forms of stress and maintain cell survival. Fas, a membrane-bound protein, is a central pro-apoptotic factor. Its activation leads to a cascade of events resulting in programmed cell death. Herein, these two mechanisms with contrary functions, promoting either cell survival or death, were addressed for their potential to inhibit each other's activation. Induction of Fas-mediated signalling was followed by a rapid decrease of HSF1 DNA binding and inducible hsp70 expression. Inhibition of HSF1 DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS-signalling. These effects of Fas-activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1)-inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase of apoptosis rates from 20% to 50% in response to heat stress. When analyzing Fas-mediated apoptosis in the presence of HSF1/hsp70 activation, decreased apoptosis rates were detected with induced expression of hsp70 but not with activation of HSF1-DNA binding alone. Thus, we conclude that inhibition of the HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis.

Animals↗
PubMed1999

Electrophysiological correlates of direct versus indirect semantic priming in normal volunteers.

The N400 component of event related potentials (ERP) was studied in 27 right-handed healthy subjects in a speeded lexical decision task. The semantic distance between primes (always words) and targets (50% words, 50% non-words) was systematically varied. Prime-target relations included directly related words (hen-egg), indirectly related words (lemon-sweet), and non-related words (sofa-wing). ERPs were recorded from 20 scalp electrodes positioned according to the international 10-20 system. The N400 reflected semantic distance with the most negative and latest N400 peak in the non-related condition, the least negative and earliest N400 peak in the direct condition and the peak in the indirect condition in-between. Hence, N400 priming effect in the indirect condition was obtained in the absence of strong sentential constraints and even though the mediating word was physically not present.

Adult↗
PubMed1999

Negative regulation of oligodendrocyte differentiation by galactosphingolipids.

Galactocerebroside and sulfatide, major galactosphingolipid components of oligodendrocyte plasma membranes and myelin, are first expressed at a critical point, when progenitors cease to proliferate and commence terminal differentiation. We showed previously that an antibody to galactocerebroside/sulfatide arrested terminal differentiation, suggesting a role for these galactolipids in oligodendrocyte differentiation. We have now investigated the differentiation of oligodendrocytes (1) in response to other anti-galactolipid antibodies, showing that anti-sulfatide O4 but not anti-galactocerebroside O1 blocks terminal differentiation, perhaps by mimicking an endogenous ligand, and (2) in a transgenic mouse unable to synthesize these lipids because of mutation of the gene for ceramide galactosyltransferase, a key enzyme for galactosphingolipid synthesis. We find that galactosyltransferase mRNA expression begins at the late progenitor [pro-oligodendroblast (Pro-OL)] stage of the lineage and that the late progenitor marker pro-oligodendroblast antigen is not synthesized in the absence of galactosyltransferase. The principal outcome of the elimination of these galactolipids is a two- to threefold enhancement in the number of terminally differentiated oligodendrocytes both in culture and in vivo. Because the general pattern of differentiation and the level of progenitor proliferation and survival appear to be unaltered in the mutant cultures, we conclude that the increased number of oligodendrocytes is caused by an increased rate and probability of differentiation. In agreement with these two experimental approaches, we present a model in which galactosphingolipids (in particular galactocerebroside and/or sulfatide) act as sensors and/or transmitters of environmental information, interacting with endogenous ligands to function as negative regulators of oligodendrocyte differentiation, monitoring the timely progress of Pro-OLs into terminally differentiating, myelin-producing oligodendrocytes.

Animals↗
PubMed1999

Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia

Hewett JA, Hewett SJ, Winkler S, Pfeiffer SE. 1999. Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia. J Neurosci Res 56:189-198. In the article referenced above, the LPS concentration employed in all studies was 1 &mgr;g/ml, not 1 mg/ml as published. This correction appears: in the Materials and Methods section on page 190, column 1, line 47; in the Results section on page 191, column 2, legend to Figure 1, line 3; on page 192, column 2, legend to Figure 2, line 3; on page 194, column 2, legend to Figure 5, line 4; on page 195, column 1, legend to Figure 6, line 3; and in the Discussion section on page 196, column 1, line 9. The publisher regrets this error.

Journal Article↗
PubMed1999

Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia.

Expression of inducible nitric oxide (NO) synthase (NOS-2) occurs during inflammation in the central nervous system (CNS) and has been linked to demyelination accompanying certain CNS inflammatory diseases. Although astrocytes and microglia are thought to be the major sources of NOS-2 expression in the CNS in vivo, recent evidence suggested that the myelin-producing oligodendrocytes (OLs) themselves can express NOS-2 in culture. Given the potentially important pathological implications of this finding, the purpose of this study was to examine further the expression of NOS-2 by OLs in vitro. After exposure to lipopolysaccharide (LPS) and interferon-gamma (IFNgamma), primary cultures enriched for mature OLs released NO in a time-dependent manner, although the amount varied considerably between different culture preparations. Increased NO production was accompanied by expression of NOS-2 mRNA and protein, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Immunofluorescence analysis revealed that the cell-type expressing NOS-2 in these cultures was galactocerebroside (Gal C)-negative but CD11b-positive. Further, NO production could be attenuated in cultures treated with the microglial/macrophage toxin, leucine methyl ester, prior to LPS/IFNgamma stimulation. Thus, microglia were the source of NOS-2 catalytic activity in these cultures. The present results indicate that LPS and IFNgamma are not effective stimuli for induction of NOS-2 in OLs in primary cell culture.

Animals↗
PubMed1999

Six3 overexpression initiates the formation of ectopic retina.

The homeobox gene sine oculis (so) is essential for visual system formation in Drosophila. A vertebrate member of the so/Six gene family, Six3, is expressed in the developing eye and forebrain. Injection of Six3 RNA into medaka fish embryos causes ectopic Pax6 and Rx2 expression in midbrain and cerebellum, resulting in the formation of ectopic retinal primordia. Injected mouse Six3 RNA initiates ectopic expression of endogenous medaka Six3, uncovering a feedback control of Six3 expression. Initiation of ectopic retina formation reveals a pivotal role for Six3 in vertebrate retina development and hints at a conserved regulatory network underlying vertebrate and invertebrate eye development.

Animals↗
PubMed1999

Improved mouth guard design for protection and comfort.

Mouth guards worn during contact sports have been shown to provide considerable protection against traumatic injuries to the teeth and supporting structures. Of the 3 types available, the custom-made mouth guard is considered superior to stock and mouth-formed mouth guards. The procedure for making a special mouth guard designed for protection and comfort is described.

Acrylic Resins↗
PubMed1999

Frequency of cytokine-producing T cells in patients of different age groups with Plasmodium falciparum malaria.

The frequency of cytokine-producing T cells was assessed in patients of different age groups (29 infants, aged 1-5 years; 30 schoolchildren, aged 6-14 years; 26 adults, aged >15 years) with acute Plasmodium falciparum malaria from Gabon. By using flow cytometry for the intracellular detection of cytokines, a striking expansion was seen, in adults compared with children, of CD4+ and CD8+ T cells with the following profiles of type 1 cytokine production: interleukin (IL)-2-/interferon (IFN)-gamma+, IL-2+/IFN-gamma+, and IL-2+/IFN-gamma-. Patients with hyperparasitemia had a significantly lower frequency of IL-2-/IFN-gamma+ CD4+ cells. Type 2 cytokine expression (IL-4+/IFN-gamma-, IL-13+/IFN-gamma-) and type 0 cytokine expression (IL-4+/IFN-gamma+, IL-13+/IFN-gamma+) were also increased in adults within the CD4+ subset. Frequencies of IL-5+/IL-4+, IL-10+/IFN-gamma-, and IL-10+/IFN-gamma+ cells were similar in all groups. The increased frequency of both type 1 and type 2 cytokine-producing T cells in adults is likely to be of significance in the protection against P. falciparum malaria.

Acute Disease↗
PubMed1999

A 6-year experience demonstrates the utility of screening for both cytogenetic and FMR-1 abnormalities in patients with mental retardation.

Cytogenetic abnormalities were found in 81 of 2,757 cases (2.93%) screened for both cytogenetic and FMR-1 mutations because of mental retardation from 1992 to 1997. Of these, 38 (46.9%) were unbalanced autosomal abnormalities, 23 (28.4%) sex chromosomal abnormalities, and 20 (24.7%) balanced autosomal rearrangements. Five subtle deletions were found, of which three involved the long arm of chromosome 7 (7q22-q31.1). Although a high-resolution banding method was used for screening, we concluded that a banding level of 450-550, generally achieved in routine analysis, was sufficient to detect all of these abnormalities. Fragile-X DNA studies revealed CGG expansion mutations in 72 other cases (2.61%). Of the abnormals, 36 (50%) were males and 8 (11.1%) females with full mutations, and 12 (16.7%) were males and 16 (22.2%) females with premutations. Together, our cytogenetic and DNA screening gave 5.5% abnormal results. In this report, these findings are compared with similar surveys of 3,940 subjects from five previous studies. From the combined data of 6,697 cases, this testing approach should reveal nearly equal numbers of cytogenetic and fragile X abnormalities in approximately 6-7% of subjects. It is productive, cost-effective, and consistent with American College of Medical Genetics (ACMG) guidelines for screening of mentally handicapped individuals.

Blotting, Southern↗
PubMed1999

Activation of Fas inhibits heat-induced activation of HSF1 and up-regulation of hsp70.

Activation of heat shock factor (HSF) 1-DNA binding and inducible heat shock protein (hsp) 70 (also called hsp72) expression enables cells to resist various forms of stress and survive. Fas, a membrane-bound protein, is a central proapoptotic factor; its activation leads to a cascade of events, resulting in programmed cell death. These two mechanisms with contradictory functions, promoting either cell survival or death, were examined for their potential to inhibit each other's activation. Induction of FAS-mediated signaling was followed by a rapid decrease in HSF1-DNA binding and inducible hsp70 expression. Inhibition of HSF1-DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS signaling. These effects of FAS activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1) inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase in apoptosis rates from 20% to 50% in response to heat stress. When analyzing the effects of HSF1/hsp70 activation on Fas-mediated apoptosis, protection from apoptosis was seen in cells with induced hsp70 protein levels, but not in cells that were just induced for HSF1-DNA binding. Thus, we conclude that inhibition of HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis, without the influence of inhibitory signals.

Apoptosis↗