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Biomedical subjects

R M Hoffman

Publications and source records attributed to R M Hoffman.

At least 91 records · Page 5Linked to original sources
PubMed1998

High expression, purification, and properties of recombinant homocysteine alpha, gamma-lyase.

Homocysteine alpha,gamma-lyase from the anaerobic protozoan parasite Trichomonas vaginalis has been cloned from genomic DNA using PCR methods and expressed in Escherichia coli with a vector containing the T7 promoter. The recombinant homocysteine alpha,gamma-lyase (rHCYase) is expressed as the major protein in the host E. coli cells. The enzyme was purified to approximately 90% purity using heat treatment at 50 degreesC, precipitation steps with polyethyleneimine, polyethylene glycol 8000, and high sodium chloride, DEAE-Sepharose FF chromatography, and phenyl-Sepharose 6 FF chromatography. The final yield was greater than 50%, which encompassed an approximate 18-fold purification. The enzyme is a homotetramer with a monomer molecular weight of 43K and contains pyridoxal phosphate. The Trichomonas rHCYase is selective for homocysteine with respect to very low cysteinase activity in contrast to the alpha,gamma-lyase from Pseudomonas putida, which has very high cysteinase activity with respect to homocysteine. The T. vaginalis and P. putida alpha,gamma-lyases readily separate on a phenyl-Sepharose 6 FF column with the T. vaginalis enzyme eluting first. rHCYase is stable up to 50 degreesC and active over a pH range of 6-8. These properties of high recombinant expression in E. coli, a simple and effective high-yield purification procedure and high relative specificity for homocysteine with respect to cysteine, make rHCYase a promising candidate to use for the diagnosis of hyperhomocystenemia, which has been demonstrated to be a major risk factor for the onset and mortality of cardiovascular disease of all types.

Animals↗
PubMed1998

A patient-like orthotopic implantation nude mouse model of highly metastatic human ovarian cancer.

Clinically relevant animal models of human cancer are important for studies of cancer biology, invasion and metastasis, and for investigating new forms of prognostic diagnosis and therapy. An ovarian tumor line (RMG-1: human clear cell carcinoma of the ovary) previously grown subcutaneously was implanted orthotopically as intact tissue into the ovarian capsule of 22 nude mice. The tumors showed progressive growth at the orthotopic site in all animals. Tumor-associated serum galactosyltransferase (GAT) tended to be positive in all nude -mice. The tumors invaded or metastasized to the contralateral ovary, retroperitoneum, mesentery and peritoneum, and omentum, and metastasized to the subcutaneous tissue, lymph nodes and distant organs including the liver, kidney, pancreas, and diaphragm. In striking contrast, subcutaneous transplantation of this tumor resulted in growth in only 2 of 5 animals with local lymph node and kidney involvement but no retroperitoneal or peritoneal involvement. These findings suggest that orthotopic implantation provides a suitable micro-environment in which ovarian cancer can express its intrinsic clinically-relevant properties. This approach is relevant to the clinical features of ovarian cancer and is thought to be a useful model for studies of therapy for this cancer.

Adenocarcinoma, Clear Cell↗
PubMed1998

Prostate-specific antigen testing practices and outcomes.

OBJECTIVES: To characterize prostate-specific antigen (PSA) testing practices in a hospital-based primary care clinic, and to determine the outcomes of PSA testing, including urology referrals, biopsies, cancers detected, and cancer treatments. DESIGN: Retrospective cohort study. Data were obtained from computerized Department of Veterans Affairs (VA) files and the statewide New Mexico Surveillance, Epidemiology, and End Results (SEER) tumor registry. SETTING: Primary care clinics in a university-affiliated VA Medical Center. PATIENTS: Subjects were 1,448 men without cancer undergoing PSA testing in 1993 with follow-up through 1994. MAIN RESULTS: Twenty-one percent of clinic enrollees at least 40 years of age were tested with PSA, including 58 who were 75 years of age or older. By the end of 1994, 40.0% (95% confidence interval [CI] 37.2%, 42.8%) were retested; 25.6% (95% CI 21.7%, 29.5%) of initial retesting occurred within 6 months. Overall, 20.7% (95% CI 18.9%, 22.5%) of PSA tests in the cohort were for men aged 75 years or older and were repeated within 6 months. Among the 193 subjects with PSA values > or = 4.0 ng/mL, 86.0% (95% CI 81.1%, 90.9%) were followed-up in urology clinic, and 46.1% (95% CI 39.1%, 53.1%) underwent biopsy. Only 11 of 51 men aged 75 years or older who were referred to urologists for an elevated PSA underwent biopsy. Forty cancers were diagnosed-a detection rate of 2.8% (95% CI 2.0%, 3.6%). Of these, 28 were organ-confined, 7 had regional invasion, and 3 had distant metastases. CONCLUSIONS: Primary care providers frequently ordered PSA tests, but a substantial proportion of testing occurred outside recommended age ranges and screening intervals. Older patients with elevated PSA values often did not complete diagnostic workups. Better adherence to screening guidelines may limit the number of both PSA tests and urology referrals.

Adult↗
PubMed1998

Topical liposome targeting of dyes, melanins, genes, and proteins selectively to hair follicles.

For therapeutic and cosmetic modification of hair, we have developed a hair-follicle-selective macromolecule and small molecule targeting system with topical application of phosphatidylcholine-based liposomes. Liposome-entrapped melanins, proteins, genes, and small-molecules have been selectively targeted to the hair follicle and hair shafts of mice. Liposomal delivery of these molecules is time dependent. Negligible amounts of delivered molecules enter the dermis, epidermis, or bloodstream thereby demonstrating selective follicle delivery. Naked molecules are trapped in the stratum corneum and are unable to enter the follicle. The potential of the hair-follicle liposome delivery system for therapeutic use for hair disease as well as for cosmesis has been demonstrated in 3-dimensional histoculture of hair-growing skin and mouse in vivo models. Topical liposome selective delivery to hair follicles has demonstrated the ability to color hair with melanin, the delivery of the active lac-Z gene to hair matrix cells and delivery of proteins as well. Liposome-targeting of molecules to hair follicles has also been achieved in human scalp in histoculture. Liposomes thus have high potential in selective hair follicle targeting of large and small molecules, including genes, opening the field of gene therapy and other molecular therapy of the hair process to restore hair growth, physiologically restore or alter hair pigment, and to prevent or accelerate hair loss.

Alopecia↗
PubMed1997

Governing step of metastasis visualized in vitro.

Metastasis is the ultimate life-threatening stage of cancer. The lack of accurate model systems thwarted studies of the metastatic cell's basic biology. To follow continuously the succeeding stages of metastatic colony growth, we heritably labeled cells from the human lung adenocarcinoma cell line ANIP 973 with green fluorescent protein (GFP) by transfection with GFP cDNA. Labeled cells were then injected intravenously into nude mice, where, by 7 days, they formed brilliantly fluorescing metastatic colonies on mouse lung [Chishima, T., Miyagi, Y., Wang, X., Yang, M., Tan, Y., Shimada, H., Moossa, A. R. & Hoffman, R. M. (1997) Clin. Exp. Metastasis 15, 547-552]. The seeded lung tissue was then excised and incubated in the three-dimensional sponge-gel-matrix-supported histoculture that maintained the critical features of progressive in vivo tumor colonization while allowing continuous access for measurement and manipulation. Tumor progression was continuously visualized by GFP fluorescence in the same individual cultures over a 52-day period, during which the tumors spread throughout the lung. Histoculture tumor colonization was selective for lung cancer cells to grow on lung tissue, because no growth occurred on histocultured mouse liver tissue, which was also observed in vivo. The ability to support selective organ colonization in histoculture and visualize tumor progression by GFP fluorescence allows the in vitro study of the governing processes of metastasis [Kuo, T.-H., Kubota, T., Watanbe, M., Furukawa, T., Teramoto, T., Ishibiki, K., Kitajima, M., Moossa, A. R., Penman, S. & Hoffman, R. M. (1995) Proc. Natl. Acad. Sci. USA 92, 12085-12089]. The results presented here provide significant, new opportunities to understand and to develop treatments that prevent and possibly reverse metastasis.

Adenocarcinoma↗
PubMed1997

Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression.

We report the establishment of stable, high-level green fluorescent protein (GFP)-expressing cell lines in vitro that permit the detection and visualization of distant micrometastases when they are implanted orthotopically in nude mice. Chinese hamster ovary cells were transfected with the dicistronic expression vector containing the humanized GFP cDNA. A stable GFP-expressing clone was selected in 1.5 microM methotrexate in vitro and injected s.c. in nude mice. Stable high-level expression of GFP was maintained in the s.c. growing tumors. To use GFP expression for metastasis studies, fragments of s.c. growing tumor, which are comprised of GFP-expressing cells, were implanted by surgical orthotopic implantation in the ovary of nude mice. Subsequent micrometastases were detected in systemic organs and could be visualized by GFP fluorescence in the lung, liver, and other organs down to the single-cell level. With this fluorescent tool, we detected and visualized for the first time tumor cells at the microscopic level in fresh viable tissue in their normal host organ. Confocal microscopy further enabled us to study physiologically relevant patterns of invasion and micrometastasis.

Animals↗
PubMed1997

Interstitial lung disease in an adult with Fanconi anemia: clues to the pathogenesis.

We have studied a 38-year-old man with a prior diagnosis of Holt-Oram syndrome, who presented with diabetes mellitus. He had recently taken prednisone for idiopathic interstitial lung disease and trimethoprim-sulfamethoxazole for sinusitis. Thrombocytopenia progressed to pancytopenia. The patient had skeletal, cardiac, renal, cutaneous, endocrine, hepatic, neurologic, and hematologic manifestations of Fanconi anemia (FA). Chest radiographs showed increased interstitial markings at age 25, dyspnea began in his late 20s, and he stopped smoking at age 32. At age 38, computerized tomography showed bilateral upper lobe fibrosis, lower lobe honeycombing, and bronchiectasis. Pulmonary function tests, compromised at age 29, showed a moderately severe obstructive and restrictive pattern by age 38. Serum alpha-1 antitrypsin level was 224 (normal 85-213) mg/dL and PI phenotype was M1. Karyotype was 46,XY with a marked increase in chromosome aberrations induced in vitro by diepoxybutane. The early onset and degree of pulmonary disease in this patient cannot be fully explained by environmental or known genetic causes. The International Fanconi Anemia Registry (IFAR) contains no example of a similar pulmonary presentation. Gene-environment (ecogenetic) interactions in FA seem evident in the final phenotype. The pathogenic mechanism of lung involvement in FA may relate to oxidative injury and cytokine anomalies.

Adult↗
PubMed1997

Overexpression and large-scale production of recombinant L-methionine-alpha-deamino-gamma-mercaptomethane-lyase for novel anticancer therapy.

The goal of the next generation of cancer chemotherapy is effective tumor-selectivity. A tumor-selective target with high therapeutic potential is the elevated methionine requirement of tumor cells relative to normal cells. We have termed the elevated requirement for methionine in tumors methionine dependence. To selectively target the methionine dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine alpha-deamino-gamma-mercaptomethane-lyase (methioninase, METase) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). The METase gene was then ligated into the pT7-7 overexpression plasmid containing the T7 RNA polymerase promoter and recloned in E. coli strain BL21(DE3). The pAC-1 clone was isolated by its yellow-orange color which is due to high enrichment of the pyridoxal phosphate-containing recombinant methioninase (rMETase) and distinguished rMETase-overproducer from rMETase-negative colonies. A scale-up production protocol which contained a heat step, two DEAE Sepharose FF ion-exchange, and one ActiClean Etox endotoxin-affinity chromatography columns has been established. The pAC-1 clone produces rMETase at approximately 10% of the total soluble protein and up to 1 g/liter in shake-flask culture. The protocol can produce therapeutic rMETase at the multi-gram level per batch with high yield (> 60%), high purity (> 98%), high stability, and low endotoxin. Purified rMETase is stable to lyophilization. The t1/2 of rMETase was 2 h when rMETase was administered by i.v. injection in mice. Studies of the antitumor efficacy of rMETase in vitro and in vivo on human tumors xenografted in nude mice demonstrated that all types of human tumors tested including those from lung, colon, kidney, brain, prostate, and melanoma were sensitive to rMETase. In contrast, normal cells were insensitive to rMETase in vitro and correspondingly, no toxicity was detected in vivo at the effective doses. In conclusion, the overexpression clone and large-scale production protocols for rMETase have enabled rMETase to be used as a tumor-selective therapeutic with broad indication and high promise for effective, low-toxicity human cancer therapy.

Amino Acid Sequence↗
PubMed1997

Topical liposome delivery of molecules to hair follicles in mice.

The hair cycle consisting of growing and resting phases, is subject to widespread disease such as androgenic alopecia or loss of pigment which are in need of effective, targeted therapeutics. In order to develop a hair-follicle delivery system we demonstrate here that phosphatidylcholine liposomes entrapping either the fluorescent dye calcein or the pigment melanin can deliver these molecules into the hair follicle and hair shafts of mice when applied topically. Liposomal delivery of these molecules is time dependent. Negligible amounts of delivered molecules enter the dermis, epidermis or blood stream thereby demonstrating the enrichment of follicle delivery. Naked calcein and melanin are trapped in the stratum corneum and are unable to enter the follicle. The potential of the hair-follicle liposome delivery system for therapeutic use for hair disease is discussed.

Administration, Topical↗
PubMed1997

Metastatic patterns of lung cancer visualized live and in process by green fluorescence protein expression.

We demonstrate here the visualization of human lung cancer metastasis live and in process in nude mice by green fluorescent protein (GFP) expression. The human lung adenocarcinoma cell line Anip 973 stably transfected with the humanized GFP-S65T cDNA was selected for very bright green fluorescence. GFP-transfected lung cancer cells were initially inoculated subcutaneously in nude mice. Five weeks after transplantation, the resulting tumor had reached over 1 cm in diameter and had very bright GFP fluorescence. Fragments of subcutaneous tumor were implanted onto the visceral pleura of the left lung of nude mice by surgical orthotopic implantation (SOI) of histologically-intact tissue via transverse thoracotomy. The ipsilateral resulting tumor was highly fluorescent due to GFP expression. GFP expression allowed the visualization of the advancing margin of the ipsilateral tumor into the fresh normal lung tissue. Lymphogenous and direct-seeding metastases in the pulmonary hilum, cervical lymph nodes, the mediastinum and contralateral pleural cavity and contralateral lung in the SOI-treated mice were brightly visualized by GFP expression in fresh tissue. GFP-transfected and untransfected tumor had similar metastatic characteristics suggesting that GFP expression had no effect on metastasis itself. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance in the visualization and study of lung cancer metastasis in process.

Adenocarcinoma↗
PubMed1997

Conversion of highly malignant colon cancer from an aggressive to a controlled disease by oral administration of a metalloproteinase inhibitor.

In this study, we describe the activity of CT1746, an orally-active synthetic MMP inhibitor that has a greater specificity for gelatinase A, gelatinase B and stromelysin than for interstitial collagenase and matrilysin, in a nude mouse model that better mimics the clinical development of human colon cancer. The model is constructed by surgical orthotopic implantation (SOI) of histologically-intact tissue of the metastatic human colon tumor cell line Co-3. Animals were gavaged with CT1746 twice a day at 100 mg/kg for 5 days after the SOI of Co-3 for 43 days. In this model CT1746 significantly prolonged the median survival time of the tumor-bearing animals from 51 to 78 days. Significant efficacy of CT1746 was observed on primary tumor growth (32% reduction in mean tumor area at day 36), total spread and metastasis (6/20 treated animals had no detectable spread and metastasis at autopsy compared to 100% incidence of secondaries in control groups). Efficacy of CT1746 could also be seen on reducing tumor spread and metastasis to individual organ sites such as the abdominal wall, cecum and lymph nodes compared to vehicle and untreated controls. We conclude that chronic administration of a peptidomimetic MMP inhibitor via the oral route is feasible and results in inhibition of solid tumor growth, spread and metastasis with increase in survival in this model of human cancer, thus converting aggressive cancer to a more controlled indolent disease.

Amides↗
PubMed1997

Methioninase: a therapeutic for diseases related to altered methionine metabolism and transmethylation: cancer, heart disease, obesity, aging, and Parkinson's disease.

Methionine metabolism and transmethylation are central to the metabolism and differentiation of all known cells. In enkaryotic organisms, methionine metabolism and transmethylation are of paramount importance in modification and regulation of proteins, lipids, and nucleic acids. The differential methylation of genes regulates their expression in the myriad of cells in eukaryotic organisms. Disruption and abnormalities in methionine metabolism and transmethylation seems to be associated with the major diseases of mankind, including cancer, heart disease, aging, obesity, and Parkinson's disease. In this review, we describe how aberrant and abnormal methionine metabolism and transmethylation are related to these major diseases. Most importantly, we review and hypothesize how the developing therapeutic recombination methioninase (rMETase) can be utilized to cure or prevent all of these diseases.

Aging↗
PubMed1996

Depth-targeted efficient gene delivery and expression in the skin by pulsed electric fields: an approach to gene therapy of skin aging and other diseases.

The ability to target genes to the various layers, cell types, and appendages of the skin could be used to correct disorders, including those of aging such as wrinkling, as well as utilize specific cell types for production molecules useful elsewhere in the body. However, the stratum corneum acts as a significant physical barrier to gene transfer into the skin. In this report we describe the ability to target and express the lacZ reporter gene to various depths of the dermis region in hairless mice. Skin-depth targeting is achieved by varying pulsed electrical fields and subsequent pressure from caliper-type electrodes on topically applied naked lacZ gene constructs. With electric pulses and extended pressure, the maximum depth of lacZ expression in the dermis and transfected cells was achieved at 370 micron and 457 cells/mm2, respectively. Gene expression was observed only the hair follicles in the case of the control.

Animals↗