Biomedical subjects

G Steinberg

Publications and source records attributed to G Steinberg.

At least 19 recordsLinked to original sources

PubMed2001

Synthesis and evaluation of pteroic acid-conjugated nitroheterocyclic phosphoramidates as folate receptor-targeted alkylating agents.

A novel nitroheterocyclic bis(haloethyl)phosphoramidate prodrug linked through lysine to a pteroic acid has been prepared and evaluated as a potential alkylating agent to target tumor cells that overexpress the folate receptor. The prodrug exhibited IC(50) values in the micromolar range and was 10-400-fold less cytotoxic in vitro than the phosphoramidate that lacks the lysine-pteroyl moiety. The data does not support a contribution of the folate receptor to cytotoxicity. In an attempt to determine the basis for the decreased cytotoxicity in the pteroyl-lysyl analogue, compounds were prepared in which the lysine-pteroyl moiety was replaced with lysine alone or with an n-propyl group. The n-propyl and the lysyl analogues were on average 3.8- and 21-fold less potent than the unsubstituted bis(haloethyl)phosphoramidate, respectively. Chemical reduction of the prodrugs followed by (31)P NMR kinetics demonstrated that all of the phosphoramidate anions cyclized to the aziridinium ion at similar rates and gave comparable product distributions, suggesting that changes in chemical activation did not account for the differences in cytotoxicity. It is likely that folate receptor-mediated transport is not sufficient to deliver adequate intracellular concentrations of the cytotoxic phosphoramide mustard.

Antineoplastic Agents, Alkylating↗

PubMed2001

Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity.

Many fungal pathogens undergo a yeast-hyphal transition during their pathogenic development that requires rearrangement of the cytoskeleton, followed by directed membrane traffic towards the growth region. The role of microtubules and their dynamic behavior during this process is not well understood. Here we set out to elucidate the organization, cellular role and in vivo dynamics of microtubules in the dimorphic phytopathogen Ustilago maydis. Hyphae and unbudded yeast-like cells of U. maydis contain bundles of spindle pole body-independent microtubules. At the onset of bud formation two spherical tubulin structures focus microtubules towards the growth region, suggesting that they support polar growth in G(2), while spindle pole body-nucleated astral microtubules participate in nuclear migration in M and early G(1). Conditional mutants of an essential alpha-tubulin gene from U. maydis, tub1, confirmed a role for interphase microtubules in determination of cell polarity and growth. Observation of GFP-Tub1 fusion protein revealed that spindle pole body-independent and astral microtubules are dynamic, with elongation and shrinkage rates comparable to those found in vertebrate systems. In addition, very fast depolymerization was measured within microtubule bundles. Unexpectedly, interphase microtubules underwent bending and rapid translocations within the cell, suggesting that unknown motor activities participate in microtubule organization in U. maydis. Movies available on-line: http://www.biologists.com/JCS/movies/jcs1792.html

Base Sequence↗

PubMed2000

A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis.

We identified a temperature-sensitive mutant of the plant pathogenic fungus Ustilago maydis that is defective in the polar distribution of cell wall components and shows abnormal morphology. The affected gene, yup1, was cloned by complementation. It encodes a putative target soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (t-SNARE), suggesting a function in membrane fusion. A Yup1-GFP fusion protein localized to vesicles that showed rapid saltatory motion along microtubules. These vesicles are part of the endocytic pathway and accumulate at sites of active growth, thereby supporting the expansion of the hyphal tip. In yup1(ts) cells, endocytosis is impaired and accumulation of Yup1-carrying endosomes at cell poles is abolished, resulting in apolar distribution of wall components and morphological alterations. This suggests that a membrane recycling process via early endosomes supports polar growth of U. maydis.

Amino Acid Sequence↗

PubMed2000

The cellular roles of molecular motors in fungi.

Motors are molecular machines that move their cargo along F-actin or microtubules. Fungal representatives of myosin, kinesin and dynein motors support many cellular processes including polar growth, cell division and mitosis. Recent progress in understanding their cellular roles has revealed common principles. However, it has become obvious that fungi have also developed diverse strategies to cope with long-distance organelle transport.

Endocytosis↗

PubMed2000

Efficacy and safety of valrubicin for the treatment of Bacillus Calmette-Guerin refractory carcinoma in situ of the bladder. The Valrubicin Study Group.

PURPOSE: We assess the efficacy and safety of intravesical valrubicin for the treatment of carcinoma in situ in patients with failure or recurrence after bacillus Calmette-Guerin (BCG) and who otherwise would have undergone cystectomy. Total anthracycline recovery in urine samples obtained within 24 hours of valrubicin administration was assessed in a subset of patients. MATERIALS AND METHODS: A total of 90 patients with recurrent carcinoma in situ after failed multiple prior courses of intravesical therapy, including at least 1 course of BCG, participated in this open label, noncomparative study. Each patient received 6 weekly instillations of 800 mg. intravesical valrubicin. Disease evaluations were made at baseline and 3-month intervals following treatment. Evaluations included cystoscopy with biopsy and urine cytology. Toxicity was noted throughout treatment and followup. No evidence of disease recurrence for 6 months or greater was considered a complete response. RESULTS: Of 90 patients 19 (21%) had a complete response, including 7 who remained disease-free at the last evaluation, with a median followup of 30 months. Additionally, 14 patients who did not meet the strict protocol definition of complete response had superficial Ta disease only. Median time to failure and/or last followup for complete responders was greater than 18 months. Recurrence has been noted in 79 patients to date, including only 2 with clinically advanced disease (stage T2). Of these 79 patients 44 (56%, 4 responders and 40 nonresponders) underwent radical cystectomy. Of the 41 patients with known pathological stage 6 (15%) had stage pT3 or greater at cystectomy. Four patients died of bladder cancer during the median followup of 30 months, none of whom was a complete responder or underwent cystectomy following valrubicin. The main side effects of valrubicin therapy were reversible local bladder symptoms. CONCLUSIONS: Valrubicin was effective and well tolerated in patients with carcinoma in situ of the bladder refractory to BCG therapy. Delaying cystectomy while attempting salvage therapy with valrubicin does not pose an undue risk to most patients.

Adjuvants, Immunologic↗

PubMed1999

Mechanisms of hyphal tip growth: tube dwelling amebae revisited.

Over 100 years ago, Reinhardt suggested that hyphal tip growth is comparable to ameboid movement inside a tube; the apical cytoplasm being protruded like a pseudopodium with the wall assembled on its surface. There are increasing data from hyphae which are explicable by this model. Fungi produce pseudopodia-like structures and their cytoplasm contains all of the major components implicated in pseudopodium production in animal cells. Most of these components are concentrated in hyphal tips and tip growth involves actin, a major component of pseudopodia. Together these data indicate that the essence of the ameboid model is still tenable. However, detailed mechanisms of tip growth remain too poorly known to provide definitive proof of the model and the behavior of the trailing cytoplasm indicates differences which are probably a response to the walled lifestyle.

Amoeba↗

PubMed1999

Intrahepatic hemorrhage after use of low-molecular-weight heparin for total hip arthroplasty.

Low-molecular-weight heparin (LMWH) has become a popular agent for prophylaxis against deep vein thrombosis and thromboembolic disease after total joint arthroplasty. LMWH allows for consistent dosing in postoperative patients without the need for laboratory monitoring. Hemorrhage is an uncommon but documented adverse reaction when using LMWH; however, intrahepatic hemorrhage has not been previously reported in conjunction with LMWH therapy. We report the case of a woman who suffered intrahepatic hemorrhage presenting with acute abdominal pain and vomiting after the use of enoxaparin for total hip arthroplasty.

Aged↗

PubMed1998

Organelle transport and molecular motors in fungi

Polarized growth, secretion of exoenzymes, organelle inheritance, and organelle positioning require vectorial transport along cytoskeletal elements. The discovery of molecular motors and intensive studies on their biological function during the past 3 years confirmed a central role of these mechanoenzymes in morphogenesis and development of yeasts and filamentous fungi. Saccharomyces cerevisiae proved to be an excellent model system, in which the complete set of molecular motors is presumed to be known. Genetic studies combined with cell biological methods revealed unexpected functional relationships between these motors and has greatly improved our understanding of nuclear migration, exocytosis, and endocytosis in yeasts. Tip growth of elongated hyphae, compared to budding, however, does require vectorial transport over long distances. The identification of ubiquitous motors that are not present in yeast indicates that studies on filamentous fungi might be helpful to elucidate the role of motors in long-distance organelle transport within higher eukaryotic cells. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Effects of the myosin inhibitor 2,3-butanedione monoxime on the physiology of fission yeast.

F-actin and associated myosins are thought to take part in a wide range of cellular processes, like motility and contraction, polarized growth, and secretion. The reagent 2,3-butanedione monoxime (BDM) is a well characterized inhibitor of the contraction of vertebrate muscle that reversibly affects myosin function and influences the intracellular concentration of Ca2+. Here we describe the influence of BDM on growth and division of the fission yeast Schizosaccharomyces pombe. At concentrations from 1-30 mM, BDM gradually inhibited formation and growth of S. pombe colonies on agar plates, with a lethal effect at > or = 15 mM. In strains of S. pombe that were blocked by elevated temperature from entry into mitosis, drug treatment reversibly decreased microtubule-independent tip growth and septation, with an IC50 value around 12 mM; nuclear division, on the other hand, was essentially unaffected by up to 15 mM BDM. At 30 mM BDM the secretion of invertase, which required both F-actin and microtubules, was decreased to the same extent as that seen when cytochalasin D was used to disrupt F-actin. However, the actin cytoskeleton was insensitive to up to 10 mM BDM, while the actin patches lost their polar distribution at 20-30 mM BDM. Cells treated with 5-20 mM BDM for 3 hours and then high pressure frozen did not show an accumulation of secretory vesicles. However, 10 mM BDM treatment disorganized the fungal cell wall, resulting in some unusually thick parts lying next to regions were the wall was almost absent. These defects could be rescued by incubating the cells in inhibitors of glucanases. Osmolytic stabilization with sorbitol rescued the effect of 15 mM BDM on colony survival, indicating that the secretion of wall components and/or wall-modifying enzymes may be the principal reason for cell death caused by BDM. Our results are consistent with the hypothesis that BDM influences actin-dependent processes in fission yeast and that actomyosin-dependent motility contributes to the secretory process of tip growth.

Actins↗

PubMed1998

Kinesin from the plant pathogenic fungus Ustilago maydis is involved in vacuole formation and cytoplasmic migration.

A gene encoding the heavy chain of conventional kinesin (kin2) has recently been identified in the dimorphic fungus Ustilago maydis (Lehmler et al., 1997). From the phenotype of kin2 null-mutants it was concluded that Kin2 might be involved in vesicle traffic towards the tip. However, this model did not explain why kin2-null mutant hyphae were unable to create empty cell compartments that are normally left behind the growing tip cell. Here we present a re-investigation of the function of Kin2 in hyphae and sporidia. We provide evidence that suggests a different and unexpected role of this kinesin motor in hyphal growth of Ustilago maydis. In addition, Kin2 was partially purified from U. maydis and in vitro properties were investigated. Isolated kinesin supported in vitro microtubule gliding at speeds of up to 1.8 micron/second, and showed motility properties and hydrodynamic behavior similar to those described for kinesin from N. crassa. It appears to be the product of the kin2 gene. Compared with wild-type sporidia, the kin2-null mutant sporidia grew normally but were defective in accumulation of Lucifer Yellow in their vacuoles, which were smaller than normal and often misplaced. The dikaryotic hyphae, produced by the fusion of two kin2-null sporidia, showed tip growth, but unlike wild-type hyphae, these structures lacked the large, basal vacuole and contain significantly more 200-400 nm vesicles scattered over the hole hypha. This defect was accompanied by a failure to generate regular empty cell compartments that are left behind in wild-type tip cells as the hyphae grow longer. These results suggest that Kin2 is a microtubule-dependent motor enzyme which is involved in the formation of vacuoles. The accumulation of these vacuoles at the basal end of the tip cell might be crucial for the formation of the empty sections and supports cytoplasmic migration during the growth of dikaryotic hyphae.

Cytoplasm↗

PubMed1998

Radiation induced renal cell cancer: a report of 4 cases and review of the literature.

PURPOSE: During clinical care in the University of Chicago renal cell carcinoma program there was 1 case of prior radiation therapy for a neuroblastoma (patient age 16 months). After this index case was found our investigation was conducted to identify other cases with apparent radiation therapy induced renal cell carcinoma and review the literature on the subject. MATERIALS AND METHODS: The clinical summaries of 573 cases from the University of Chicago renal cell carcinoma program from 1992 through 1998 were reviewed. RESULTS: Four cases (3 men and 1 woman) of renal cell carcinoma, including the index case, were identified with prior therapeutic radiation therapy. Of the patients 3, 32, 35 and 36 years old, had been radiated for neuroblastoma 31, 32 and 35 years, respectively, before diagnosis of renal cell carcinoma and 1 (age 64 years) had been radiated for testicular seminoma 25 years earlier. The 32-year-old patient had 2 primary renal cell carcinomas in the remaining kidney and an associated leiomyosarcoma. He was alive and disease-free on dialysis 8 years after diagnosis. The 35-year-old patient had localized well differentiated renal cell carcinoma and was disease-free for 24 months. The 36-year-old patient had relapse 7 years after nephrectomy and had progressive refractory disease 12 years after diagnosis. The 64-year-old patient had relapse in 2.5 years but was alive with metastatic disease 4 years later. A literature review documented 4 other patients with renal cell carcinoma of those with neuroblastoma treated with radiation therapy. CONCLUSIONS: Radiation therapy induced renal cell carcinoma is rare (less than 1% of renal cell carcinoma cases), and in our 4 cases it occurred 25 to 35 years after radiation. The risk of renal cell carcinoma should be considered during long-term followup of patients exposed to radiation therapy.

Adult↗

PubMed1997

Identification of a motor protein required for filamentous growth in Ustilago maydis.

The phytopathogenic fungus Ustilago maydis exists in two stages, the yeast-like haploid form and the filamentous dikaryon. Both pathogenicity and dimorphism are genetically controlled by two mating-type loci, with only the filamentous stage being pathogenic on corn. We have identified two genes (kin1 and kin2) encoding motor proteins of the kinesin family. Kin1 is most similar to the human CENP-E gene product, while Kin2 is most closely related to the conventional kinesin Nkin of Neurospora crassa. Deletion mutants of kin1 had no discernible phenotype; delta kin2 mutants, however, were severely affected in hyphal extension and pathogenicity. The wild-type dikaryon showed rapid tip growth, with all the cytoplasm being moved to the tip compartment. Left behind are septate cell wall tubes devoid of cytoplasm. In delta kin2 mutants, dikaryotic cells were formed after cell fusion, but these hyphal structures remained short and filled with cytoplasm. A functional green fluorescent protein (GFP)-Kin2 fusion was generated and used to determine the localization of the motor protein by fluorescence microscopy. Inspection of the hyphal tips by electron microscopy revealed a characteristic accumulation of darkly stained vesicles which was absent in mutant cells. We suggest that the motor protein Kin2 is involved in organizing this specialized growth zone at the hyphal tip, probably by affecting the vectorial transport of vesicles.

Amino Acid Sequence↗

PubMed1997

Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa.

Kinesin is a force-generating molecule that is thought to translocate organelles along microtubules, but its precise cellular function is still unclear. To determine the role of kinesin in vivo, we have generated a kinesin-deficient strain in the simple cell system Neurospora crassa. Null cells exhibit severe alterations in cell morphogenesis, notably hyphal extension, morphology and branching. Surprisingly, the movement of organelles visualized by video microscopy is hardly affected, but apical hyphae fail to establish a Spitzenkörper, an assemblage of secretory vesicles intimately linked to cell elongation and morphogenesis in Neurospora and other filamentous fungi. As cell morphogenesis depends on polarized secretion, our findings demonstrate that a step in the secretory pathway leading to cell shape determination and cell elongation cannot tolerate a loss of kinesin function. The defect is suggested to affect the transport of small, secretory vesicles to the site involved in protrusive activity, resulting in the uncoordinated insertion of new cell wall material over much of the cell surface. These observations have implications for the presumptive function of kinesin in more complex cell systems.

Biological Transport↗

PubMed1997

A kinesin-like mechanoenzyme from the zygomycete Syncephalastrum racemosum shares biochemical similarities with conventional kinesin from Neurospora crassa.

The kinesin superfamily consists of mechanoenzymes that convert chemical energy, stored in nucleoside triphosphates, into movement along microtubules. The founding member of this protein superfamily, the so-called conventional kinesin, was only known from animal sources until the recent description of kinesin from the filamentous fungus Neurospora crassa (G. Steinberg, M. Schliwa, Mol. Biol. Cell 6, 1605-1618 (1995)). To determine whether similar motors with comparable features are common in other filamentous fungi, a kinesin from a zygomycete, Syncephalastrum racemosum, was purified. Here, the isolation and characterization of this motorenzyme is described. The purified protein consisted of a doublet at 112 kDa and 115 kDa with no additional polypeptides. This was consistent with a calculated molecular mass of approximately 240 kDa and suggests that the motor is a dimer with a more globular shape than conventional kinesin from animal sources. In gliding assays the enzyme moved microtubules at 2.5 to 3.4 microns/s and had a nucleotide specificity similar to the Neurospora kinesin motor. Peptide antibodies against conserved regions in the head and the tall domain of conventional kinesins cross-reacted with the Syncephalastrum motor. In vitro, the enzyme was able to drive the microtubule-dependent movement of vesicles isolated from Syncephalastrum racemosum, as well as Neurospora crassa, and Aspergillus nidulans. In summary, the Syncephalastrum motor has many of the unique features in common with the conventional kinesin from the ascomycete Neurospora crassa and probably shares a similar function in living hyphae.

Animals↗

PubMed1996

A family intervention to delay nursing home placement of patients with Alzheimer disease. A randomized controlled trial.

OBJECTIVE: To determine the long-term effectiveness of comprehensive support and counseling for spouse-caregivers and families in postponing or preventing nursing home placement of patients with Alzheimer disease (AD). DESIGN: Randomized controlled intervention study. SETTING: Outpatient research clinic in the New York City metropolitan area. PARTICIPANTS: Referred, volunteer sample of 206 spouse-caregivers of AD patients who enrolled in the study during a 3 1/2-year period. All patients were living at home at baseline and had at least 1 relative living in the area. INTERVENTION: Caregivers in the treatment group were provided with 6 sessions of individual and family counseling within 4 months of enrollment in the study and were required to join support groups. In addition, counselors were available for further counseling at any time. MAIN OUTCOME MEASURE: Time from enrollment of caregivers in the study to placement of the AD patients in a nursing home. RESULTS: Using Kaplan-Meier survival analysis, we estimated that the median time (weighted average of estimates for male and female caregivers) from baseline to nursing home placement of AD patients was 329 days longer in the treatment group than in the control group (z=2.29; P=.02). The relative risk (RR) from a Cox proportional hazard model of nursing home placement (intent-to-treat estimate) after adjusting for caregiver sex, patient age, and patient income was 0.65 (95% confidence interval [CI], 0.45 to 0.94; P=.02), indicating that caregivers were approximately two thirds as likely to place their spouses in nursing homes at any point in time if they were in the treatment group than if they were in the control group. Treatment had the greatest effect on risk of placement for patients who were mildly demented (RR, 0.18; 95% CI, 0.04 to 0.77) or moderately demented (RR, 0.38; 95% CI, 0.17 to 0.82). CONCLUSIONS: A program of counseling and support can substantially increase the time spouse-caregivers are able to care for AD patients at home, particularly during the early to middle stages of dementia when nursing home placement is generally least appropriate.

Aged↗

PubMed1996

Characterization of the biophysical and motility properties of kinesin from the fungus Neurospora crassa.

Neurospora kinesin (Nkin) is a distant relative of the family of conventional kinesins, members of which have been identified in various animal species. As in its animal counterparts, Nkin most likely is an organelle motor. Because it is a functional homologue of the kinesin heavy chain of higher eukaryotes, its biophysical and motility properties were compared with those of other conventional kinesins. Purified Nkin behaves as a homodimeric complex composed of two subunits of a 105-kDa polypeptide. Based on its hydrodynamic properties (Stokes radius and sedimentation coefficient), Nkin is an elongated molecule, although it is more compact than its animal counterparts. A detailed comparison of the motility properties of Nkin with those of animal conventional kinesins reveals similarities and some intriguing differences. Nkin is less effective than other kinesins in the use of natural nucleoside triphosphates but responds to a selection of ATP analogues in a similar fashion as mammalian kinesin. Even in the presence of saturating concentrations of ATP, Nkin is significantly more sensitive to ADP or tripolyphosphate than other kinesins. Both the ATP-driven microtubule gliding activity and the microtubule-stimulated ATPase activity of Nkin obey Michaelis-Menten kinetics. Surprisingly, however, the Km values for both these activities are approximately an order of magnitude higher than those of other kinesins. Whether the low affinity for ATP suggested by these high Km values is related to the high rate of motility remains to be determined.

Adenosine Triphosphatases↗

PubMed1996

Overview of methodologic issues for pharmacologic trials in mild, moderate, and severe Alzheimer's disease.

To address the issue of mild, moderate, and severe Alzheimer's disease (AD), it is necessary to initially establish some agreement on terminology. In recent decades, these terms have frequently been defined using screening instrument scores with measures such as the Mini-Mental State Examination (MMSE). There are many problems with this approach, perhaps the most salient of which is that it has contributed to the total and tragic neglect of patients with severe AD. An alternative approach to the classification of AD severity is staging. This approach has advanced to the point where moderately severe and severe AD can be described in detail. Procedures for describing this previously neglected latter portion of AD have recently been extensively validated. Staging is also uniquely useful at the other end of the severity spectrum, in differentiating early aging brain/behavior changes, incipient AD, and mild AD. Temporally, with staging procedures, it is possible to track the course of AD approximately three times more accurately than with the MMSE. The net result of the advances in AD delineation is that issues such as prophylaxis, modification of course, treatment of behavioral disturbances, loss of ambulation, progressive rigidity, and the development of contractures in AD patients can now be addressed in a scientifically meaningful way that will hopefully bestow much benefit in AD patients and those who care for them.

Activities of Daily Living↗

PubMed1996

Mortality and temporal course of probable Alzheimer's disease: a 5-year prospective study.

Alzheimer's disease (AD) is associated with an increased mortality in comparison with aged control populations. The relationship between the clinical and the temporal course of AD has not been well studied over significant intervals. Community-residing patients with probable AD (N = 103, 42 men, mean age = 70.2 +/- 8.0 years) were studied at baseline on demographic and clinical variables, including measures of global deterioration (Global Deterioration Scale; GDS), mental status and cognition (e.g., Mini-Mental State Examination; MMSE), and functional impairment (Functional Assessment Staging; FAST). Baseline characteristics included a GDS range of Stage 4, 5, or 6 (38.8%, 39.8%, and 21.4%, respectively) and a mean MMSE score of 15.4 +/- 5.6. The mean follow-up interval was 4.6 +/- 1.4 years. Follow-ups were done blind to baseline measures and when necessary were conducted in residential and nursing home settings. Of locatable subjects (n = 95, 92%), 30 (31.6%) were decreased. Survivors (n = 65) had a mean GDS stage of 6.2 +/- 0.9 and a mean MMSE score of 5.1 +/- 6.9; 51% had MMSE scores of 0. Increased age and male gender, but not baseline clinical dementia variables, increased the risk of death (ps < .01). Change in clinical variables correlated significantly with time elapsed (r = .32, p < .05, for MMSE change, to r = .48, p < .001, for GDS change). Significant variance in temporal change (i.e., time elapsed) was accounted for by change in two of the five clinical measures studied (i.e., GDS and FAST; multiple r = .53). The results support previous estimates of mean duration of the GDS and FAST stages. For subjects with probable AD followed over approximately 5 years, clinical variables changed significantly over time in survivors. However, the majority of temporal variance in the course of AD remains unexplained.

Aged↗