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M Horwitz

Publications and source records attributed to M Horwitz.

At least 37 records · Page 2Linked to original sources
PubMed1997

On the mechanism of DNA transfection: efficient gene transfer without viruses.

From an investigation of how transfected DNA navigates from the cell surface to the nucleus, we have developed a transfection method for primary human fibroblasts that approaches the efficiency of viruses. We have visually tracked the subcellular routing of exogenous DNA and find that all cells in an asynchronous population are surprisingly competent in the nuclear uptake of DNA, but two steps practically limit efficient transfection to a minority of cells. First, regardless of the method used to traverse the cell membrane--CaPO4 precipitation, lipofection or electroporation--it appears that nuclear transport of DNA requires routing through endosomes and lysosomes. Apparent abrogation of endosome-lysosome fusion or translocation with microfilament or microtubule toxins, respectively, inhibits the nuclear accumulation of transfected DNA, but interruption of lysosomal function with protease inhibitors promotes it. Second, in normal human fibroblasts, which are refractory to transfection the exogenous DNA is rapidly excluded from the nucleus, but in HeLa cells, which are readily transfected, there is prolonged nuclear stability of the DNA, indicating the failure in HeLa cells of a mechanism for the elimination of foreign DNA. These observations imply strategies for optimizing gene transfer efficiency in virus-independent approaches to gene therapy.

Cysteine Proteinase Inhibitors↗
PubMed1997

The genetics of familial leukemia.

Familial leukemia is rare, but, as is the case with other cancer family syndromes, its study is likely to lead to the identification of genes causative of the far more common, sporadic cases. I review the clinical and, what is known of the molecular genetic features of familial leukemia. I propose a nosology based on whether the leukemia is a component of a medical syndrome or exists as a solitary disease, the apparent mode of inheritance, and the distribution of leukemia types and subtypes in affected family members. I review the recent findings from my group that leukemia is inherited with 'anticipation', in the form of a declining age of onset with each passing generation. I consider two models of leukemia genesis that can potentially account for anticipation in familial cases and incorporate epidemiological observations made in sporadic cases. The first model is analogous to trinucleotide repeat expansion in Huntington disease, myotonic dystrophy, and other inherited neurodegenerative illness demonstrating anticipation. The second model considers evidence that anticipation may be common to multiple types of familial cancer and is based on the intergenerational inheritance of multiple downstream mutations resulting from a defect in a single DNA repair gene.

Ataxia Telangiectasia↗
PubMed1997

Genetic heterogeneity in familial acute myelogenous leukemia: evidence for a second locus at chromosome 16q21-23.2.

The identification of genes responsible for the rare cases of familial leukemia may afford insight into the mechanism underlying the more common sporadic occurrences. Here we test a single family with 11 relevant meioses transmitting autosomal dominant acute myelogenous leukemia (AML) and myelodysplasia for linkage to three potential candidate loci. In a different family with inherited AML, linkage to chromosome 21q22.1-22.2 was recently reported; we exclude linkage to 21q22.1-22.2, demonstrating that familial AML is a heterogeneous disease. After reviewing familial leukemia and observing anticipation in the form of a declining age of onset with each generation, we had proposed 9p21-22 and 16q22 as additional candidate loci. Whereas linkage to 9p21-22 can be excluded, the finding of a maximum two-point LOD score of 2.82 with the microsatellite marker D16S522 at a recombination fraction theta = 0 provides evidence supporting linkage to 16q22. Haplotype analysis reveals a 23.5-cM (17.9-Mb) commonly inherited region among all affected family members extending from D16S451 to D16S289. In order to extract maximum linkage information with missing individuals, incomplete informativeness with individual markers in this interval, and possible deviance from strict autosomal dominant inheritance, we performed nonparametric linkage analysis (NPL) and found a maximum NPL statistic corresponding to a P-value of .00098, close to the maximum conditional probability of linkage expected for a pedigree with this structure. Mutational analysis in this region specifically excludes expansion of the AT-rich minisatellite repeat FRA16B fragile site and the CAG trinucleotide repeat in the E2F-4 transcription factor. The "repeat expansion detection" method, capable of detecting dynamic mutation associated with anticipation, more generally excludes large CAG repeat expansion as a cause of leukemia in this family.

Chromosome Mapping↗
PubMed1996

Long term correction of bilirubin-UDP-glucuronosyltransferase deficiency in Gunn rats by administration of a recombinant adenovirus during the neonatal period.

Injection of a recombinant adenovirus expressing human bilirubin-UGT1 (Ad-hBUGT1) (3 x 10(9) plaque-forming units (pfu) intravenously) in adult bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats resulted in biliary excretion of bilirubin glucuronides and a 70% reduction of serum bilirubin levels. However, the effect was transient, and host humoral and cellular immune response prevented transgene expression after subsequent injections. To determine whether injection during the neonatal period would tolerize the host to the recombinant virus, we injected 1 x 10(8) pfu of Ad-hBUGT1 or Ad-LacZ (a recombinant adenovirus expressing Escherichia coli beta-galactosidase) into 1-3-day-old Gunn rats. Two subsequent injections (3 x 10(9) pfu) were given 56 and 112 days after the initial injection. Injection of Ad-BUGT1, but not Ad-LacZ, reduced serum bilirubin by 70-76% of the levels in untreated pups (9 +/- 1.3 mg/dl), followed by a gradual increase to 3.25 +/- 0.3 mg/dl in 56 days; similar or greater reductions occurred after the second and third injection. Serum neutralizing antibody titer and cytotoxic lymphocyte activity against adenovirus-infected hepatocytes were low or undetectable. Thus, tolerization by injection of the virus during the neonatal period permits long term gene therapy by repeated injection of the virus.

Adenoviridae↗
PubMed1996

Hypermethylated myoblasts specifically deficient in MyoD autoactivation as a consequence of instability of MyoD.

MyoD is one of a family of basic helix loop helix (bHLH) transcription factors acting as master switches of skeletal muscle differentiation. In addition to transcriptionally activating differentiation-specific genes, it autoactivates its own expression through a positive feedback loop. It was cloned following the observation that treatment with the DNA methylation inhibitor 5-azacytidine converts cultured fibroblasts into muscle, presumably through the activation of a transcriptionally silenced locus. In an attempt to experimentally recapitulate this phenomenon, I have stably transfected mouse C2C12 myoblasts with a selectable marker fused to the MyoD promoter/enhancer, treated the cells with methyldeoxycytidine triphosphate to promote genomic hypermethylation, and negatively selected for loss of activity from the MyoD promoter/enhancer. Several clones were recovered that had lost expression of MyoD and the ability to differentiate to myotubes, but could be reverted by treatment with 5-azacytidine. One clone ("C2G2") was studied in detail. C2G2 cells resume differentiation through the forced expression of exogenous MyoD, but paradoxically fail to autoactivate the endogenous MyoD, suggesting that they are deficient in the MyoD positive feedback circuit but otherwise competent in downstream events in myogenesis. The myogenic bHLH proteins in this clone exhibit nuclear instability. Both the nuclear stability of MyoD and resumption of the autoactivation circuit may be restored either through cell fusion with fibroblasts or by treatment of the cells with protease inhibitors. This suggests that the transcriptional silencing of a factor, not in itself necessary for muscle differentiation but governing the proteolytic stability of MyoD, is responsible for the decay of the autoactivation circuit in these cells. A theoretical analysis offers an explanation for how increased rates of MyoD turnover may result in the kinetic dissociation of autoactivation from the "cross-activation" of downstream genes and suggests a mechanism for the phenomenon of myogenic "memory."

3T3 Cells↗
PubMed1996

A family inheriting different subtypes of acute myelogenous leukemia.

Rare inherited cancer syndromes have proven invaluable for the identification of genes involved in the more frequent corresponding noninherited cases. We report on a family with an adult onset, incompletely penetrant, autosomal dominant syndrome of myelodysplasia and acute myelogenous leukemia, affecting at least eight, and probably ten, individuals from three generations. The patients have developed leukemias differing in morphologic subtype, tumor cytogenetics, and abruptness of presentation. Some have presented with acute onset and others with protracted myelodysplasia. This family does not have an unusual incidence of other malignancies; however, one person at 50% risk of inheriting this gene developed atypical mycobacterium infection in the absence of leukemia, but also without appreciable risk factors for acquired deficiencies in cellular immunity. Features common to affected family members, including the individual with mycobacterium infection, are the early presence in the bone marrow of red cell and platelet maturation defects. A search for mutations in diseased marrows fails to detect abnormalities of p53 or N-ras. Two of the affected family members, third degree relatives, have co-inherited a constitutional chromosomal banding variation of 9p21-22, potentially suggesting linkage to this locus. The variable penetrance and expressivity of this syndrome support a multistep model of leukemia evolution, in which the gene defined by this family's syndrome is the signal step.

Adolescent↗
PubMed1996

Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

Animals↗
PubMed1996

Preferential MyoD homodimer formation demonstrated by a general method of dominant negative mutation employing fusion with a lysosomal protease.

We report on a general strategy for engineering dominant negative mutations that, in principle, requires neither extensive structural or functional knowledge of the targeted protein. The approach consists of fusing the lysosomal protease cathepsin B (CB) to a subunit of a multimeric protein. The CB fusion polypeptide can proteolytically digest the multimer and/or detour the multimer from its usual subcellular destination to the lysosome. We first demonstrate the general validity of the approach with CB fusion to E. coli lacZ, encoding tetrameric beta-galactosidase. Cotransfection of NIH 3T3 cells with a vector expressing a CB-lacZ fusion inhibits the beta-galactosidase activity produced by transfection of lacZ alone. We infer that the dominant negative inhibition results from both direct proteolysis of the beta-galactosidase tetramer by the fusion subunit and detour of the tetramer to the lysosome. In a specific application of this strategy, we have fused CB to the dimeric bHLH skeletal muscle transcription factor MyoD. The CB-MyoD fusion protein localizes to the cytoplasm, presumably the lysosome, demonstrating the dominance of lysosomal localization to nuclear localization. The CB-MyoD fusion appears to divert homodimerizing native MyoD from its usual nuclear destination, consequently inhibiting MyoD-mediated transactivation and in vitro differentiation of C2C12 myoblasts. Surprisingly, the CB-MyoD fusion fails to interact with the bHLH heterodimerization partners, E12 and E47, suggesting preferential MyoD homodimer formation, at least in the prenuclear cellular compartments.

3T3 Cells↗
PubMed1996

Use of wet mount to predict Chlamydia trachomatis and Neisseria gonorrhea cervicitis in primary care.

BACKGROUND AND OBJECTIVES: Cervicitis is associated with salpingitis, infertility, and complications of pregnancy. Universal screening has been recommended for high-prevalence populations but may not be appropriate in the family practice setting. Leukocytes on an endocervical gram stain have been associated with infectious cervicitis due to Chlamydia trachomatis and Neisseria gonorrhea. This study sought to determine whether the finding of leukocytes in a vaginal wet mount could be used to screen for infectious cervicitis in an urban family practice. METHODS: A consecutive sample of 357 women had cultures for C trachomatis and N gonorrhea and a standardized wet mount. RESULTS: All women with infectious cervicitis were under age 35. Thirty-six percent of infected women had more leukocytes than epithelial cells in the wet mount, compared with 23% of women without these organisms. CONCLUSIONS: Wet mount findings did not reliably predict infectious cervicitis. Study of a larger population is needed to confirm these findings.

Adolescent↗
PubMed1996

Anticipation in familial leukemia.

Anticipation refers to worsening severity or earlier age at onset with each generation for an inherited disease and primarily has been described for neurodegenerative illnesses resulting from expansion of trinucleotide repeats. We have tested for evidence of anticipation in familial leukemia. Of 49 affected individuals in nine families transmitting autosomal dominant acute myelogenous leukemia (AML), the mean age at onset is 57 years in the grandparental generation, 32 years in the parental generation, and 13 years in the youngest generation (P < .001). Of 21 parent-child pairs with AML, 19 show younger ages at onset in the child and demonstrate a mean decline in age at onset of 28 years (P < .001). Of 18 affected individuals from seven pedigrees with autosomal dominant chronic lymphocytic leukemia (CLL), the mean age at onset in the parental generation is 66 years versus 51 years in the youngest generation (P = .008). Of nine parent-child pairs with CLL, eight show younger ages at onset in the child and reveal a mean decline in age at onset of 21 years (P = .001). Inspection of rare pedigrees transmitting acute lymphocytic leukemia, chronic myelogenous leukemia, multiple types of leukemia, and lymphoma is also compatible with anticipation. Sampling bias is unlikely to explain these findings. This suggests that dynamic mutation of unstable DNA sequence repeats could be a common mechanism of inherited hematopoietic malignancy with implications for the role of somatic mutation in the more frequent sporadic cases. We speculate on three possible candidate genes for familial leukemia with anticipation: a locus on 21q22.1-22.2, CBL2 on 11q23.3, and CBFB or a nearby gene on 16q22.

Adolescent↗
PubMed1995

A data-generated basis for medical ethics education: categorizing issues experienced by students during clinical training.

PURPOSE: To use issues identified by students in order to establish an experience- and evidence-based approach to medical ethics education. METHOD: A total of 628 sophomore and senior students at the State University of New York at Buffalo School of Medicine and Biomedical Sciences were asked to identify incidents during their clinical training that had raised ethical concerns. The sophomores were surveyed during two time periods: 1979-80, and 1991-92 and 1992-93; the seniors were surveyed in 1991-92 and 1992-93. Responses were analyzed and categorized through content analysis. RESULTS: In all, 249 students (45% of the sophomores and 20% of the seniors) responded. The categories of issues identified were professional norms, limits of intervention, defensive shielding of professional colleagues, respect toward patients, communication, and student boundaries (situations where the student feels uncomfortable). The most frequently reported incidents reflected the students' perceptions of lapses in level of care (under- or over-treatment), communication, respect toward patients, and maintenance of professional norms. The seniors and the 1979-80 sophomores reported respect toward patients as an issue less often than did the 1991-92 and 1992-93 sophomores. The seniors most often identified concerns raised over limits of intervention and resource allocation. CONCLUSION: The differences between the responses of the sophomores and seniors tend to support other research suggesting a retardation of moral sensitivity in the course of medical education. It may be that clinical teaching and faculty behavior model values at odds with what is taught in the classroom. Ethics education should focus on issues relevant to students' experience.

Ambulatory Care↗
PubMed1994

Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I.

Efficient initiation by the DNA polymerase of adenovirus type 2 requires nuclear factor I (NFI), a cellular sequence-specific transcription factor. Three functions of NFI--dimerization, DNA binding, and activation of DNA replication--are colocalized within the N-terminal portion of the protein. To define more precisely the role of NFI in viral DNA replication, a series of site-directed mutations within the N-terminal domain have been generated, thus allowing the separation of all three functions contained within this region. Impairment of the dimerization function prevents sequence-specific DNA binding and in turn abolishes the NFI-mediated activation of DNA replication. NFI DNA-binding activity, although necessary, is not sufficient to activate the initiation of adenovirus replication. A distinct class of NFI mutations that abolish the recruitment of the viral DNA polymerase to the origin also prevent the activation of replication. Thus, a direct interaction of NFI with the viral DNA polymerase complex is required to form a stable and active preinitiation complex on the origin and is responsible for the activation of replication by NFI.

Adenoviruses, Human↗
PubMed1994

The use of ultrasonography to scan the abdomen of patients presenting for routine physical examinations.

BACKGROUND: Ultrasonography has become an increasingly important diagnostic tool, producing high-quality images at a low cost. However, except in obstetrics, ultrasonography has not been used for screening purposes in asymptomatic persons. METHODS: This prospective study included 189 patients on whom an abdominal ultrasound scan was performed by a family physician as part of a routine physical examination. During the 2-year follow-up period, the screening was evaluated by determining whether the ultrasound findings contributed to the patient's health care management. RESULTS: Forty-two of the patients (22%) were found to have previously undiagnosed conditions. The most common findings were gallstones, urinary retention, and renal cysts. Six patients (3%) received treatment for the condition detected by the screening, but three of these patients received treatment only after they developed symptoms during the 2-year follow-up period. One patient developed symptoms for gallstones that may have been missed by the screening ultrasound. The internal and external reliability rates for the screening examination were 96% and 82%, respectively. CONCLUSIONS: Ultrasound findings altered the treatment plan for 3% of the screened patients but was the sole factor leading to treatment in only 1.6%. Abdominal ultrasound can be performed accurately and at a reasonable cost by generalist physicians. Patient acceptance was high, and many reported feeling reassured by the ultrasound screening.

Abdomen↗
PubMed1988

Measuring quality of care.

In the years ahead, defining and measuring the quality of medical care will be the major concern of health care professionals and policymakers in their effort to deliver equitable, cost-effective medical care. What follows is an exposition of the difficulties that surround the concept of "quality" as applied to medical care, and suggestions as to how we might begin to surmount these difficulties.

Data Collection↗
PubMed1988

Corporate reorganization: the last gasp or last clear chance for the tax-exempt, nonprofit hospital?

The current revolution in health care organization and financing, increased competition, and a retrenching of industry from its commitments to expansion of health care benefits challenge the nonprofit hospital's existence as a viable entity. Hospital governing boards and administrators have turned to corporate reorganization in order to maintain their financial position and to continue to serve their communities. This Article examines the not-for-profit concept and the problems facing nonprofit hospitals. It reviews the pros and cons of reorganization and the for-profit/nonprofit controversy. It questions whether the hybridization of the hospital results in a stronger or weaker species and discusses the possible effects of the newly structured entity on the quality and delivery of health care. Finally, the Article suggests that the nonprofit hospital may survive only by a continued commitment to societal and communal values, to service rather than to profit; that this commitment is adequate justificaton for the preservation of the nonprofit system, and its preservation will reinforce and strengthen the concept.

Cost Control↗
PubMed1987

Left ventricular thrombus in agnogenic myeloid metaplasia.

A 40-year-old white male with agnogenic myeloid metaplasia presented to our institution with symptoms of fever, rash and pleuropericardial pain. A two-dimensional echocardiogram revealed a pedunculated left ventricular mass which simulated a left ventricular myxoma. Left ventricular wall motion and coronary arteries were normal on preoperative angiography. The mass was surgically removed and found to be fibrin thrombus. A mild chronic inflammatory infiltrate was present in the base of the thrombus. The formation of thrombus in the left ventricle was ascribed to spontaneous aggregation of platelets and myocarditis of unknown cause.

Adult↗