Biomedical subjects
J J Steinberg
Publications and source records attributed to J J Steinberg.
Environmental ethics.
The U.S. Environmental Protection Agency (EPA) held the first meeting on environmental ethics sponsored by the Scientific Advisory Panel and Board on 10-11 December 1998 in Arlington, Virginia (1). The report from the meeting will more completely inform scientists and the community of current issues. This editorial should serve as an initial brief of this meeting [which was held on the fiftieth anniversary of the Declaration of Human Rights (adopted by the United Nations on 10 December 1948)].
Cisplatin DNA adduct detection and depurination measured by 32P DNA radiolabeling and two-dimensional thin-layer chromatography: a time and concentration study.
Platinum-based chemotherapies cause the formation of DNA adducts and have profound effects on DNA. This study measured cis-diamminedichloroplatinum II (cisplatin) DNA adducts by 32P-radiolabeling DNA, enzymatically digesting radiolabeled DNA, separating the formed adducts on two-dimensional thin-layer chromatography, and quantitating the adducts with autoradiography and densitometry. HeLa DNA was incubated with cisplatin at varying concentrations (6.25-325 nM) and times (0 min to 72 hr). Cisplatin rapidly depurinated dGMP and dAMP (90%, 15-min incubation with 325 nM cisplatin). Partial depurination of dGMP (15%) and dAMP (25%) occurred with lower cisplatin concentrations at equal incubation times. A minimum of four new adducts, with relatively rapid migratory patterns, were detected at high cisplatin concentrations with short incubation times. These results indicate that the depurination of DNA correlates with DNA adduct formation and that the quantification of these adducts may be applicable to monitoring tumor and host cell response to cisplatin chemotherapy.
Variation in the deoxynucleotide composition between organic and nonorganic strawberries.
The differences in xenobiotic chemical content between organic and nonorganic products can be chemically measured. In this study, the deoxynucleotide composition of strawberry samples is used to demonstrate chromatographic methods of quantifying the differences between pesticide- and toxic-exposed strawberries. The samples were analyzed by (32)P labeling and two-dimensional thin-layer chromatography. This technique is sensitive for detecting rare (1/10(10)) deoxynucleotide adducts and analogues (minor bases) in DNA. The results indicate differences in the amount and type of adduct formation between organic and nonorganic strawberry samples and within different parts of the same type of sample. The elucidation of the content and effects of pesticides and toxics in foods is critical in differentiating organic from common produce. It can also aid the agricultural industry in improving the application of chemicals in pest management. Furthermore, it helps to enhance the understanding of long-term epidemiology in nutrition research, especially in susceptible populations. These findings are of particular application in the pediatric population, where dietary habits are restricted to specific food groups.
The immunohistological diagnosis of E. coli O157:H7 colitis: possible association with colonic ischemia.
OBJECTIVE: E. coli O157:H7 may cause hemorrhagic colitis resembling ischemic colitis. Diagnosis is usually made by finding sorbitol-negative colonies on MacConkey agar that react with O157 and H7 antisera. Most ischemic colitis is idiopathic, but some may be caused by E. coli O157:H7, inasmuch as this organism can produce fibrin thrombi in colon vasculature. The objectives of this study were to determine whether E. coli O157:H7 infection can be diagnosed retrospectively from paraffin blocks of colon sections and whether an association exists between E. coli O157:H7 infection and colonic ischemia. METHODS: Paraffin-embedded sections of normal colon (n = 2) and various colitides [ischemic (n = 11), E. coli O157:H7 (n = 2), IBD (n = 8) and pseudomembranous (n = 3)] were used. Sections were deparaffinized, rehydrated, incubated with 3% peroxide in methanol, rinsed, and incubated with peroxidase-labeled antibody isolated from goats immunized with whole E. coli O157:H7. Sections were stained with peroxidase chromagen reagent and counterstained with hematoxylin. Coarse, granular, orange-brown staining was considered positive. To determine the localization of the chromagen deposits, three cases that stained positive, including one of the culture-proved E. coli O157:H7 colitis and two of colonic ischemia, were processed for electron microscopy. RESULTS: Both cases (100%) of E. coli O157:H7 colitis and three of 11 (27.3%) cases of ischemic colitis stained positive by light microscopy. In one culture-proved case, electron microscopy demonstrated staining of bacillary structures; in two cases of colonic ischemia, extensive deposits of chromagen material were present that were associated neither with inflammatory cells nor with bacterial forms. CONCLUSIONS: Immunoperoxidase staining of archival sections may be used to diagnose E. coli O157:H7 infection. An etiological role for this organism is possible in some cases of colonic ischemia.
Interstrain variation in the deoxynucleotide composition of Cryptococcus neoformans: nucleotide composition of Cryptococcus neoformans.
The deoxynucleotide (dNMP) composition of ten strains of C. neoformans was analysed by 32P-labelling and two-dimensional thin-layer chromatography. This technique is very sensitive for detecting rare deoxynucleotide adducts and analogues (minor bases) in DNA. The results indicate considerable variation among strains in DNA nucleotide composition.
In vivo determination of 5-bromo-2'-deoxyuridine incorporation into DNA tumor tissue by a new 32P-postlabelling thin-layer chromatographic method.
The halopyrimidine 5-bromo-2'-deoxyuridine (BUDR) can serve as one of many indicators of tumor malignity, complementary to histologic grade. We have developed a thin-layer chromatographic (TLC) technique that can assess tumor DNA base composition and analogue (BUDR) incorporation which vies with immunochemistry for BUDR. This requires post-labeling DNA by nick-translation and radioactive 5'-phosphorylation of representative 32P-alpha-dNMPs (deoxynucleotide monophosphates). Subsequent 3'-monophosphate digest exchanges a radioactive 32PO4 for the neighboring cold nucleotide. Separation in two dimensional PEI-cellulose TLC is carried out in acetic acid, (NH4)2SO4, and (NH4)HSO4. TLC of dNMPs was applied to control HeLa DNA, and HeLa cells receiving BUDR. BUDR is detected in 10(6) HeLa cells after 12-72 h incubations. Findings in HeLa DNA demonstrate normal TLC retention factors for all 32P-dNMPs. Two dimensional R(F) (x,y axes in cm) demonstrate: dAMP=1.4, 9.4; dCMP=10.0, 13.5; dGMP=4.6, 4.4; dTMP=9.0, 7.4; and BUDRMP 6.4, 6.6. This technique quantifies BUDR--which parallels tumor S phase, and serves as an indicator of labelling index (LI).
Morphologic pattern of tenascin as a diagnostic biomarker in colon cancer.
BACKGROUND: Immunohistochemical methods were used to study the pattern of expression of tenascin (TN) in invasive colon cancer and its relation to prognosis. METHODS: Sixty patients (29 males, 31 females) with a mean age of 77 years were studied. TN expression was evaluated by immunohistochemistry using paraffin-embedded tissue sections, TN expression levels were correlated with patient age, tumor stage, and survival. RESULTS: TN positivity varied from trace to 4+. Staining patterns were as follows: in well-differentiated cancer, TN fibers form thick bands around invading tumor glands. In poorly differentiated cancer, TN fibers had an interstitial pattern surrounding individual tumor cells. Using Cox's proportional hazard regression method, survival was significantly related to TN score (P < 0.0001) and stage of disease (P < 0.05). No significant relationship was found between survival and age (P = 0.375). CONCLUSION: Patients with more TN expression had better long-term survival than patients with no or weak TN expression. Pathologic and clinical entities in colon cancer have distinct immunohistochemical TN matrix patterns that may correlate with predictive value and long-term survival.
Contrasting effects of identical nutrients given parenterally or enterally after 70% hepatectomy: bacterial translocation.
High mortality occurs in rats with 70% hepatectomy fed intravenous (IV) total parenteral nutrition (TPN; 13.9% glucose, 4.17% amino acids, 1.46% fat, electrolytes, trace minerals, and vitamins providing 216 kcal.kg-1.d-1) but not when the identical nutrients are given at the same rate enterally (gastrostomy). We hypothesized that a difference in bacterial translocation (BT) was a contributing factor to this phenomenon. Forty-five male Sprague-Dawley rats (300-360 g) were divided into five groups and underwent the following: control (no operation), sham (intraperitoneal [IP] pentobarbital anesthesia, central venous and gastrostomy catheters, laparotomy, sham hepatectomy), standard oral feeding (SOF), TPN (IV nutrients), and total enteral nutrition (TEN; gastrostomy). The SOF, TPN, and TEN groups had IP pentobarbital anesthesia, central venous and gastrostomy catheters, and 70% hepatectomy. Postoperatively, control and SOF (both catheters plugged) rats ate a commercial rat chow and drank tap water ad libitum pre- and postoperatively. The sham, TPN, and TEN groups were given the identical infusate composition as above, but the nutrient concentrations were cut in half (110 kcal/kg) and three-quarters (165 kcal/kg) on postoperative days 1 and 2, respectively. At the end of postoperative day 2, all rats were euthanized. BT to mesenteric lymph nodes (MLNs), liver, spleen, and lungs was significantly higher in the TPN rats compared with all other groups, except that BT to the MLNs was similar in the TPN and TEN groups. Bacteremia was found only in the TPN rats. BT in TPN rats with 70% hepatectomy was significantly greater 48 h after operation than in those fed the identical nutrients enterally at the same rate; this correlates with the previously reported significantly greater mortality in rats with 70% hepatectomy receiving TPN.
The formation and measurement of DNA neuroadduction in alcoholism. Case report.
We present a case report of an intoxicated alcoholic driver who sustained fatal motor vehicle injuries. We subsequently quantified ethanol-derived acetaldehyde (ACE) DNA products in his brain, which may represent a major contributor to clinical alcoholic use and complications. Further, ACE DNA neuroadducts may indicate chronic exposure to alcohol, as demonstrated by 32P-prelabeled DNA and two-dimensional thin-layer chromatography. ACE and other unknown neuroadducts were evident in the histologically normal frontal, parietal, and caudate lobes. DNA neuroadduct formation was extensive and similar in three separate brain regions with normal histology. Contributing neuroadduction by chronic drug abuse is also possible, though the deceased's terminal acute blood screens for recent drug abuse were negative. The mechanism of alcohol neurotoxicity remains unknown, but biochemical nonenzymatic changes of DNA at the nucleic acid level (adduct formation) can alter gene function and stability. DNA neuroadduct detection may represent an important determinant in quantifying neurotoxicity from drug abuse or alcoholism in the absence of history, the presence of negative blood, tissue, and urine assays for recent drug and alcohol use, and the absence of neuropathology.
Traumatic pancreatitis: method and effects of i.v. fluids and Sandostatin.
UNLABELLED: Somatostatin and its analogs are used clinically to treat patients with pancreatitis. To evaluate the effects of i.v. Sandostatin (SNST) on rats with trauma-induced acute pancreatitis, 130 male Sprague-Dawley rats (300-350 g) underwent celiotomy, controlled direct pancreas contusion, and central i.v. line insertion under ip sodium pentobarbital anesthesia. The rats were divided randomly into control (IA, IIA, and IIIA) and SNST-treated (IB, IIB, and IIIB) groups. The basic infusion solution contained 4.8% glucose, vitamins, and electrolytes. For groups IA and IB, the infusion rate was 24 ml/kg/day, while it was 240 ml/kg/day for groups IIA, IIB, IIIA, and IIIB. SNST administration was 6 micrograms/kg/hr i.v. for groups IB and IIB during the first postoperative day, while group IIIB received 6 micrograms/kg/hr i.v. for 4 days. Surviving rats were euthanized after 4 days. All survivors and nonsurvivors were autopsied. In all groups, severity of pancreatitis, fat necrosis, and ascites were greater in the nonsurvivors (P < 0.005 in each case). Mortality rates were consistently lower in the SNST groups: IA (76%) vs IB (52%), IIA (71%) vs IIB (50%), and IIIA (63%) vs IIIB (50%). Because individual group mortality rates were not affected by volume of infusate given or length of time SNST was administered, the results of all control and all SNST rats were combined; there was a statistically significant lower mortality in the SNST-treated rats (51 vs 71%, P < 0.04). CONCLUSION: Intravenous administration of Sandostatin to rats following induction of severe acute traumatic pancreatitis significantly ameliorates the course of the disease.
Overexpression of NGF within the heart of transgenic mice causes hyperinnervation, cardiac enlargement, and hyperplasia of ectopic cells.
Nerve growth factor (NGF) supports the survival of developing sympathetic and a subpopulation of sensory neurons. In the adult it participates in maintenance of the neurotransmitter phenotype of responsive neurons. The amount of NGF synthesized by a given target tissue determines its final innervation density; those developing neurons that fail to receive sufficient NGF undergo apoptosis. In order to examine the ramifications of this principle in the context of a specific target organ, a transgenic mouse model was developed in which NGF expression was increased in developing and adult cardiac tissue by placing a NGF minigene under the transcriptional control of the cardiac-specific alpha-myosin heavy chain promoter. Transgenic mice developed cardiac enlargement secondary to both an increase in myocardial mass and the presence of an abundant ectopic cell population. Immunohistochemical analyses with the neural marker S-100 revealed staining of a subpopulation of ectopic cells, suggesting their derivation from the neural crest. Whereas immunostaining for the neuronal-specific protein neuron-specific enolase demonstrated labeling of another subpopulation of ectopic cells within the heart. Measurements of cardiac tissue catecholamine levels revealed a marked elevation in transgenic mice, consistent with sympathetic hyperinnervation. Analysis of mediastinal sympathetic ganglia revealed increases in both the size and the number of neurons. In this model, increased expression of NGF produced hyperinnervation of the heart, pathological cardiac growth, and the recruitment and/or expansion of an ectopic, neural crest-derived cell type.
Contrasting effects of identical nutrients given parenterally or enterally after 70% hepatectomy.
Based on clinical observations, we hypothesized that prolonged parenteral nutrition (in contrast to enteral nutrition) is detrimental after major hepatic resection. Male Sprague-Dawley rats (300 to 380 g) anesthetized with intraperitoneal sodium pentobarbital had 70% hepatic resection and jugular vein and gastrostomy catheterizations using aseptic techniques and were divided randomly into three groups: (1) total parenteral nutrition (TPN) (nutrients via central vein), (2) total enteral nutrition (TEN) (identical nutrients via gastrostomy), and (3) standard oral feeding (SOF) (chow and water ad libitum). Unused catheters were plugged. In the first set of experiments (n = 42), nutrient intake was formulated to approximate the nutritional intake of normal rats, 216 kcal/kg/d. Infusate was 15% glucose, 4.5% amino acids, electrolytes, trace minerals, vitamins, and 20% fat emulsion given half-strength the first day, three-fourths strength the second day, and full strength thereafter. On postoperative day 7, surviving rats were killed. Mortality prior to day 7 was very high (68%) in the TPN group and low in the TEN (9%) and SOF (9%) groups (p < 0.005). Among survivors, the serum albumin level was lowest (p < 0.002) and serum bilirubin level (p < 0.025) and wet weight of regenerated liver (p < 0.002) highest in the TPN group. However, the livers in TPN rats appeared pale and were found to be abnormal histologically with markedly diminished glycogen and amphophylic hepatocyte cytoplasm, and their spleens were enlarged (by a factor of two). The high mortality of TPN rats was seen whether the fat emulsion was given as a bolus daily, continuously as part of the infusate, or not included as part of the TPN regimen. In the next series (n = 70), nutrient concentrations, volumes, and rates of infusion were varied. There was a high correlation between caloric (r2 = 0.831, p < 0.0006), glucose (r2 = 0.598, p < 0.02), and amino acid (r2 = 0.619, p < 0.03) intakes and mortality in the TPN group: at 140 kcal/kg/d, none died; at 178 kcal/kg/d, 50% to 62% died; and at 230 kcal/kg/d, 80% died. No TEN rat died. In conclusion, 70% hepatectomized rats fed enterally with nutrients approximating the intake of normal rats do well and survive. In sharp contrast, mortality is very high when identical nutrients are infused parenterally. By reducing the levels of nutrients given parenterally, survival improves significantly.
Assay of deoxy-deazapurines in DNA by 3'-phosphorylation and two-dimensional thin-layer chromatography.
Deoxy-deazapurines (deaza-dNMPs) are incorporated into cellular DNA after administration of anti-neoplastic, anti-viral, or anti-parasitic chemotherapy. Deaza-dNMPs are stable purine analogues and can be detected via 32P-labeling cold DNA. Assay of analogue incorporation and normal base composition is carried out by radiolabeling DNA with all four deoxynucleotides (dNMPs) through nick translation. 3'-Monophosphate digest radiolabels representative dNMPs and deaza-dNMPs. Separation occurs in two-dimensional polyethyleneimine-cellulose thin-layer chromatography, which resolves all dNMPs. The technique was applied to human placental and calf thymus DNA, control and altered calf thymus DNA with cold stoichiometric replacement of deaza-dNMPs to include deoxy-deazaadenosine, deoxy-deazaguanosine, and deoxy-deazainosine. Scintillation detection and densitometry both accurately reflect dNMP content. This technique easily and quickly quantifies the low-molecular-mass deaza-dNMP analogues in DNA. Deaza-dNMP uptake into DNA may reflect clinical chemotherapeutic efficacy and host toxicity. The assay may therefore serve as an early biochemical dosimeter of drug effect and resistance.
A comparison of AgNOR staining and BUDR labeling of metastatic tumors of the brain.
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Lactate turnover at rest and during submaximal exercise in patients with heart failure.
Systemic and lower limb skeletal muscle lactate metabolism was studied in 10 men with congestive heart failure by use of a primed continuous intravenous infusion of L-(+)-[U-14C]lactate. Arterial and deep femoral venous blood samples were obtained at rest and during 30 min of submaximal exercise. Systemic lactate metabolic turnover rate (Rd) was determined using Steele's isotopic steady-state equation (Rd = isotopic infusion rate/arterial specific activity). Plasma lactate concentrations in the artery and deep femoral vein did not change significantly from resting values during exercise (1.11 +/- 0.13 vs. 1.26 +/- 0.12 and 1.27 +/- 0.12 vs. 1.30 +/- 0.12 mM, respectively), whereas Rd increased from 22.5 +/- 1.8 to 41.6 +/- 4.8 mumol.kg-1.min-1 (P < 0.005). Rd did not significantly correlate with arterial lactate concentration during rest or exercise. Because of simultaneous uptake and release of lactate in skeletal muscle, arterial and deep femoral venous lactate concentrations are not closely related to either systemic or lower limb skeletal muscle lactate metabolism in patients with congestive heart failure.
Enzymatic shot-gun 5'-phosphorylation and 3'-sister phosphate exchange: a two-dimensional thin-layer chromatographic technique to measure DNA deoxynucleotide modification.
DNA adducts occur through environmental, therapeutic, dietary, oxygen stress, and aging processes. A modified thin-layer chromatographic (TLC) technique can asses base composition and adduct formation. This requires labeling DNA by "shot-gun" 5'-phosphorylation of representative 32P-alpha-deoxyribonucleotide monophosphates. Subsequent 3'-monophosphate digest "sister exchanges" a radioactive 32PO4(2-) to the neighboring cold nucleotide. Separation in two-dimensional polyethyleneimine-cellulose TLC is carried out in acetic acid, (NH4)2SO4, and (NH4)HSO4. The technique was applied to control DNA, cold substitution of dUMP, methylation, depurination, and pBR322. This technique quantifies low-molecular-mass adducts and DNA integrity both in vivo and in vitro.
Postprandial hypoglycemia in islet beta cell hyperplasia with adenomatosis of the pancreas.
Organic hyperinsulinism causing hypoglycemia in adults is caused by insulinoma, islet hyperplasia, or a combination of adenomata and hyperplasia. We present a patient with long-standing symptoms of postprandial hypoglycemia occurring within 15 minutes of meals in the absence of fasting hypoglycemic symptoms. An intravenous glucagon stimulation test resulted in a rise of plasma insulin from 194 to 21,883 pmol/L at 7.5 minutes. Blood glucose simultaneously rose from 4.9 to 5.9 mmol/L. A glucose tolerance test revealed an exuberant insulin response. A euglycemic hyperinsulinemic clamp demonstrated incomplete suppression of plasma C-peptide. At surgery, three nodules were found and a 50-60% distal pancreatectomy was performed. The pancreas revealed a combination of multiple beta-cell islet adenomata and islet hyperplasia with no evidence of nesidioblastosis. The coexistence of islet adenomata with hyperplasia must be considered in the differential diagnosis of postprandial hypoglycemia.