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D Gruber

Publications and source records attributed to D Gruber.

At least 37 records · Page 2Linked to original sources
PubMed1999

Microtubule-associated protein 4 (MAP4) regulates assembly, protomer-polymer partitioning and synthesis of tubulin in cultured cells.

We depleted MAP4, a ubiquitously expressed microtubule (MT)-associated protein previously shown to be capable of stabilizing MTs, from HeLa cells by stably expressing antisense RNA. These HeLa-AS cells, in which the MAP4 level was decreased to 33% of the wild-type level, displayed decreased content of total tubulin (65% of the wild-type level). The partitioning of cellular tubulin into protomer and polymer was altered in HeLa-AS cells: polymeric tubulin was decreased to 46% of the level in control cells, while protomeric tubulin was increased to 226% of the level in control cells. Tubulin protein synthesis was decreased, consistent with the tubulin autoregulation model, which proposes that tubulin protomer inhibits its own synthesis. Following release from drug-induced depolymerization, MTs in HeLa-AS cells reformed more slowly, and showed an increased focus on the centrosome, as compared to control cells. HeLa-AS cells also appeared to be less bipolar in shape and flatter than control cells. Our data suggest that MAP4 regulates assembly level of MTs and, perhaps through this mechanism, is involved in controlling spreading and shape of cells.

Biopolymers↗
PubMed1999

E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics.

Microtubule-associated proteins (MAPs) have been hypothesized to regulate microtubule dynamics and/or functions. To test hypotheses concerning E-MAP-115 (ensconsin) function, we prepared stable cell lines expressing conjugates in which the full-length MAP (Ensc) or its microtubule-binding domain (EMTB) was conjugated to one or more green fluorescent protein (GFP) molecules. Because both distribution and microtubule-binding properties of GFP-Ensc, GFP-EMTB, and 2x, 3x, or 4xGFP-EMTB chimeras all appeared to be identical to those of endogenous E-MAP-115 (ensconsin), we used the 2xGFP-EMTB molecule as a reporter for the behavior and microtubule-binding function of endogenous MAP. Dual wavelength time-lapse fluorescence imaging of 2xGFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimera associated with the lattice of all microtubules immediately upon polymerization and dissociated concomitant with depolymerization, suggesting that dynamics of MAP:microtubule interactions were at least as rapid as tubulin:microtubule dynamics in the polymerization reaction. Presence of both GFP-EMTB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellular microtubules at all cell cycle stages suggested that the MAP might function in modulating stability or dynamics of microtubules, a capability shown previously in transiently transfected cells. Although cells with extremely high expression levels of GFP-EMTB chimera exhibited stabilized microtubules, cells expressing four to ten times the physiological level of endogenous MAP exhibited microtubule dynamics indistinguishable from those of untransfected cells. This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likely serves to modulate microtubule functions or interactions with other cytoskeletal elements.

Female↗
PubMed1999

GFP chimeras of E-MAP-115 (ensconsin) domains mimic behavior of the endogenous protein in vitro and in vivo.

E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of chimeric proteins consisting of five GFP molecules attached to a single EMTB molecule produces brightly labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, we have demonstrated the utility of chimeric proteins containing GFP multimers as authentic reporters of ensconsin distribution and dynamics; expression of these GFP-EMTB chimeric molecules also provides a non-perturbing label of the microtubule system in living cells.

Animals↗
PubMed1999

[Non-penetrating deep sclerectomy in the surgical treatment of chronic open-angle glaucoma. Mid-term results].

PURPOSE: Non-penetrating deep sclerectomy has been performed in France since the early nineties and appears to be an interesting alternative to Cairn's trabeculectomy. The technical characteristics, the ability to use antimitotic procedures and postoperative YAG laser goniotomy contribute to make deep sclerectomy an attractive surgical method. We evaluated its efficacy and adverse effects in a mid-term retrospective series. PATIENTS AND METHODS: Fifty patients (all POAG) without usually accepted failure risks for trabeculectomy (trabeculoretraction less than 3 months, intraocular anterior or posterior lens, aphakia, black or Asian subject, failure of previous surgical procedure, patients under 40) underwent this surgical procedure between June 96 and October 97 performed by several skilled surgeons in our unit. This was the first antiglaucoma surgical procedure for all patients. Collagen draining implant was not used. Two pressure criteria (21 mmHg and 16 mmHg) were used to assess success. Success rate and adverse effects were compared with previously published data using the Kaplan-Meier test. RESULTS: Medium follow-up was 14.24 months. The success rate was 81% (IOP 21 mmHg) and 50% (IOP 16 mmHg) at maximum follow-up of 18 months. There was no statistical difference between treated and untreated groups for target IOP at 21 mmHg (p = 0.12). These results were comparable to those in previous studies and to those obtained with trabeculectomy. The complication rate was low (hyphema 0%, choroidal detachment 2%, hypothalamia 2%, endocular infection 0%). DISCUSSION: Our success rate and complication rate were comparable with previously published series. Choosing a target IOP of 16 mmHg allowed a better comparison between daily clinical observations and mid-term results, showing a significant difference from the 21 mmHg target. Nevertheless, the success rate was comparable to that obtained with trabeculectomy and the complication rate was lower, supporting the favorable opinion concerning deep sclerectomy. CONCLUSION: Non-penetrating deep sclerectomy appears to be as efficient as Cairn's trabeculectomy for surgical treatment of glaucoma and allows a lower complication rate. Long-term results, visual field and papilla remain to be evaluated. Furthermore, results with a pressure goal of 16 mmHg are interesting to evaluate because they reflect the real clinical situation better than the target 21 mmHg IOP. This technique should be evaluated in other forms of glaucoma.

Actuarial Analysis↗
PubMed1998

Reduction of intraocular pressure in a glaucoma patient undergoing hormone replacement therapy.

OBJECTIVES: To show the reducing effect of estrogens and progestins on the elevated intraocular pressure (IOP) in the case of a 56-year-old woman showing typical climacteric complaints, who was admitted to the menopause outpatient unit. She also suffered from a primary open-angle glaucoma treated with betaophtiole eye drops with intraocular pressures of 16-20 mmHg under this local therapy. METHODS: IOP patterns were monitored by means of standardised daily pressure profiles four times a day before as well as 4 and 12 weeks after the beginning of hormone replacement therapy (HRT). The local glaucoma therapy remained unchanged. RESULTS: During HRT, IOP levels were reduced from 16-20 mmHg before therapy to 12-15 mmHg at week 4 and to 13-15 mmHg at week 12 after the beginning of HRT. CONCLUSION: The finding of a close chronological relationship between the onset of menopause and the development of a glaucoma is a potentially new indication for HRT.

Antihypertensive Agents↗
PubMed1998

Impact of the age at menarche on adult body composition in healthy pre- and postmenopausal women.

The present study focuses on the impact of age at menarche on body composition development during adulthood. With 459 healthy middle-class women between 18 and 67 years (x = 41.5) the association between age at menarche and body composition was tested. Body composition, described by absolute and relative amount of fat mass, lean body mass, and bone mass, was estimated by means of dual energy x-ray absorptiometry. In order to exclude the influence of the menopausal transition on body composition, pre- and postmenopausal females were examined separately. The absolute amount of body fat was significantly lower within the group of women whose menarche occurred later. However, postmenopausal females exhibit less significant relations between the two trait systems than premenopausal women. This may be due to the impact of menopausal transition which affected the hormone levels and body composition development independently from the adolescent hormonal transition. While in both proband groups the quantitative amount of body fat was significantly related to menarcheal age, a significant relation between menarcheal age and adult body fat distribution could not be verified.

Adolescent↗
PubMed1998

Identification of kinesin-like molecules in myogenic cells.

Numerous organelles are repositioned during myogenic differentiation and are maintained in an asymmetric distribution throughout the life span of a myotube. It is likely that members of the kinesin superfamily may be responsible for some or all of these microtubule-dependent movements. Consequently, we have attempted to identify kinesin-like molecules expressed throughout myogenesis. Using a standard PCR-based strategy, we cloned two kinesin-like molecules from a rat myogenic cell line, L6. Sequence analysis of the first of these, KIF3C, defines it as a novel member of the KIF3 subfamily of kinesin-like proteins. KIF3C is expressed throughout myogenesis as well as in numerous rat tissues. Like other members of the KIF3 subfamily, KIF3C has an N-terminal motor domain. The second molecule identified is a rat homolog of murine KIF1B, a putative mitochondrial transporter. KIF1B is also expressed ubiquitously both in myogenic cells at all stages and in a variety of rat tissues.

Amino Acid Sequence↗
PubMed1998

Pruritic urticarial papules and plaques of pregnancy: clinical and immunopathologic observations in 57 patients.

BACKGROUND: Pruritic urticarial papules and plaques of pregnancy (PUPPP) has been described either as a homogeneous or a polymorphic clinical process. Its cause is unknown. OBJECTIVE: We attempted to characterize the clinical and immunopathologic findings in PUPPP on the basis of long-term clinical and immunopathologic observations. METHODS: The clinical and immunopathologic features of 57 patients with PUPPP were evaluated. RESULTS: The clinical features in 57 patients with PUPPP were categorized into three types: mainly urticarial papules and plaques (type I), nonurticarial erythema, papules, or vesicles (type II), and combinations of the two forms (type III). Direct immunofluorescence studies in 48 of the 57 patients showed nonspecific immunoreactants in dermal blood vessels and/or moderate granular deposits at the dermoepidermal junction in 15 patients. CONCLUSION: Type I PUPPP differed from types II and III in clinical appearance and distribution (absence of face, palm, and sole lesions), but trimester onset, parity, and direct immunofluorescence findings were not significantly different among the three groups.

Adolescent↗
PubMed1998

The impact of nutritional status on body fat distribution patterns in pre- and postmenopausal females.

This study examines the impact of nutritional status, classified by body mass index, on sex specific fat distribution patterns dependent on menopausal status in 467 pre-, peri- or postmenopausal females. Absolute and relative amounts of upper and lower body fat were estimated by means of dual energy X-ray absorptiometry. It was found that low weight, independent of menopausal status, leads to the typical gynoid pattern of fat distribution while excess weight and obesity result in the android pattern of distribution in pre- and postmenopausal women.

Adipose Tissue↗
PubMed1998

Treatment of menopausal keratoconjunctivitis sicca with topical oestradiol.

OBJECTIVE: To investigate the effect of 17 beta-oestradiol ophthalmic drops in comparison with a traditional tear substitute in postmenopausal women with keratoconjunctivitis sicca. DESIGN: Randomised prospective trial. SETTING: Menopause clinic. PARTICIPANTS: Eighty-four postmenopausal women suffering from keratoconjunctivitis sicca and necessitating a hormone replacement therapy (HRT) for general climacteric symptoms. METHODS: The women were randomised into two groups and were given 17 beta-oestradiol eye drops (n = 42, group 1) or a tear substitute (n = 42, group 2). Both groups received a systemic HRT. MAIN OUTCOME MEASURES: A Schirmer's test was performed immediately before the beginning of therapy and after four months. In addition, eye symptoms were assessed using a visual analogue scale. RESULTS: A comparison of visual analogue scores at four months in the women who received 17 beta-oestradiol eye drops versus those who received a tear substitute demonstrated a statistically significant difference in all observed ocular symptoms (P < 0.0001). The Schirmer's test revealed a significant difference of results before and after treatment in the oestradiol group (P < 0.0001) while in group 2 no significant difference was found. CONCLUSIONS: Our study demonstrates that topical oestrogen is successful in treating keratoconjunctivitis sicca while it seems that the blood-eye barrier prevents systemic oestrogens from acting on the conjunctivae.

Administration, Topical↗
PubMed1997

Overexpression of full- or partial-length MAP4 stabilizes microtubules and alters cell growth.

To investigate the in vivo functions of MAP4, a microtubule-associated protein expressed almost ubiquitously in vertebrate cells, we prepared stably transfected clonal mouse Ltk- cell lines expressing full-length MAP4 (L-MAP4 cells) or its MT-binding domain (L-MTB cells). Although transfectants showed no dramatic defect in morphology, organellar distribution, or level of MT polymer, as compared to naive Ltk- cells or L-MOCK cells (transfected with vector alone), MTs in L-MAP4 and L-MTB cells showed greater stability than those in control cells, as monitored by the level of post-translationally detyrosinated alpha-tubulin and by a quantitative nocodazole-resistance assay. In vivo, the MT-binding domain of MAP4 stabilized MTs less potently than full-length MAP4, in contrast to the equivalent efficacy demonstrated in studies of in vitro MT polymerization (Aizawa et al. (1991), J. Biol. Chem. 266, 9841-9846), L-MAP4 and L-MTB cells grew significantly more slowly than control cells; this growth inhibition was not due to mitotic arrest or cell death. L-MAP4 and L-MTB cells also exhibited greater tolerance to the MT-depolymerizing agent, nocodazole, but not to the MT-polymerizing agent, Taxol. Our results demonstrate that MAP4 and its MT-binding domain are capable of MT stabilization in vivo, and that increasing the intracellular level of MAP4 affects cell growth parameters.

Animals↗
PubMed1997

Overexpression of MAP4 inhibits organelle motility and trafficking in vivo.

We previously prepared cell lines that inducibly overexpress MAP4, a microtubule (MT)-associated protein widely expressed in non-neuronal cells. Overexpression of either the full-length MAP4 molecule or its MT-binding domain, MTB, stabilized MTs and retarded cell growth, suggesting that overexpressed MAP4 impacts on MT-dependent functions in vivo. To test this hypothesis, we examined MT-based vesicle movements in living cells, using high resolution DIC microscopy. Overexpression of either MAP4 or MTB yielded a dose-dependent reduction in the frequency of MT-dependent organelle movements, relative to control cells. At steady state, both MAP4- and MTB-overexpressing cells showed unusual distributions of transferrin, LDL, dextran, and Golgi elements, as compared to control cells. MAP4 preferentially inhibited receptor-dependent uptake and degradation of LDL, and repositioning of Golgi elements after disruption by the drug, brefeldin A. L-MOCK cells treated with Taxol to stabilize the MTs to an extent equivalent to MAP4 overexpression did not show similar inhibition of vesicle motility or organellar trafficking, suggesting that deficits in organelle movements in vivo represent a direct effect of the presence of MAP4 or MTB, rather than an indirect effect of the stabilization of MTs by overexpressed MAP constructs. Our results show that MAP4 has the capacity to affect transport along MTs in vivo; these findings suggest a potential mechanism by which MAP4 could contribute to polarization or morphogenesis of cells.

Biological Transport↗
PubMed1996

Measurement of amniochorionic membrane thickness using high-frequency ultrasound.

Premature rupture of the membranes (PROM) accounts for approximately 30 per cent of all preterm deliveries. PROM is thought to be mainly due to a decrease in membrane integrity. The aim of our investigation was to determine, post-partum after 28 normal deliveries, the thickness of the amniochorionic membrane using a 20 MHz high-frequency ultrasound. The data obtained were compared with histological sections for measurement accuracy using a linear regression analysis method. The membrane thickness of the total study group was 0.83 +/- 0.11 mm (0.72-1.08 mm). Based on a statistical comparison with the histological sections, the high-frequency ultrasound examination was shown to be highly reliable, with a correlation coefficient of r = 0.96 (P < 0.0001). High-frequency ultrasonographic examinations of membrane thickness are an objective and reliable method and may be a gain to prenatal diagnostics once this method can be used in vivo.

Adolescent↗
PubMed1996

Decreased sexual interest and its relationship to body build in postmenopausal women.

OBJECTIVES: The relationship between body build, androgen levels and changes in sexual interest after menopause was investigated in 171 postmenopausal women from Vienna, Austria. METHODS: All women were interviewed using a structured questionnaire. Body build was determined by employing five absolute body dimensions and four anthropometric indices. RESULTS: Body weight, as well as the amount of subcutaneous centripetal fat (such as in the chest, waist and hip region), were statistically significantly related to the degree of reduced sexual interest. Corpulent and heavy women suffered far more frequently from a severe decrease in sexual interest after menopause. Statistically significant associations between androgen levels and decrease in sexual interest could not be demonstrated. CONCLUSIONS: Reduced sexual interest is associated with a kind of body type not corresponding to the culture-specific beauty ideals of our society, first of all evident in women whose menopause occurred relatively early.

Adult↗
PubMed1995

Structural features mediating fibrin selectivity of vampire bat plasminogen activators.

The distinguishing characteristic of vampire bat (Desmodus rotundus) salivary plasminogen activators (DSPAs) is their strict requirement for fibrin as a cofactor. DSPAs consist of structural modules known from urokinase (u-PA) and tissue-type plasminogen activator (t-PA) such as finger (F), epidermal growth factor (E), kringle (K), and protease (P), combining to four genetically and biochemically distinct isoenzymes, exhibiting the formulas FEKP (DSPA alpha 1 and alpha 2) and EKP and KP (DSPA beta and DSPA gamma). Only DSPA alpha 1 and alpha 2 bind to fibrin. All DSPAs are single-chain molecules, displaying substantial amidolytic activity. In a plasminogen activation assay, all four DSPAs are almost inactive in the absence of fibrin but strongly stimulated by fibrin addition. The catalytic efficiency (kcat/Km) of DSPA alpha 1 increases 10(5)-fold, whereas the corresponding value of t-PA is only 550. The ratio of the bimolecular rate constants of plasminogen activation in the presence of fibrin versus fibrinogen (fibrin selectivity) of DSPA alpha 1, alpha 2, beta, gamma, and t-PA was found to be 13,000, 6500, 250, 90, and 72, respectively. Whereas all DSPAs are therefore more fibrin dependent and fibrin selective than t-PA, the extent depends on the respective presence of the various domains. The introduction of a plasmin-sensitive cleavage site in a position akin to the one in t-PA partially obliterates fibrin cofactor requirement. Fibrin dependence and fibrin selectivity of DSPAs are accordingly mediated by fibrin binding, which involves the F domain, as yet undefined determinants within the K and P domains, and by the absence of a plasmin-sensitive activation site. These findings transcend the current understanding of fibrin-mediated stimulation of plasminogen activation: in addition to fibrin binding, specific protein-protein interactions come into play, which stabilize the enzyme in its active conformation.

Aminocaproic Acid↗