In vitro pore-forming activity of the lantibiotic nisin. Role of protonmotive force and lipid composition

Human prostate cancer displays a high degree of variability in its rate of spread, which could be due largely to differences in the invasive potential of the tumor cells. The degradation of the basal lamina and stromal extracellular matrix is mediated in part by the secretion of matrix metalloproteinases (MMPs). Matrilysin (PUMP-1, MMP-7) and gelatinase A (M(r) 72,000 type IV collagenase, MMP-2) have been shown to be overexpressed in prostate carcinoma. We have expressed the single MMP matrilysin in the tumorigenic but nonmetastatic human prostate tumor cell line DU-145 to determine if matrilysin has a functional role in prostate tumor cell invasion. DU-145 cells expressing matrilysin were significantly more invasive than vector-only transfected cell lines as assayed by a severe combined immunodeficient mouse model of tumor cell invasion. Vector-only transfected DU-145 cells injected i.p. into severe combined immunodeficient mice invaded the diaphragm in only 1 of 9 mice (11%), whereas matrilysin-transfected DU-145 cells invaded the diaphragm in 12 of 18 mice (66%). The difference between the controls and matrilysin-transfected cells was statistically significant (P < 0.006). These results suggest a functional role for matrilysin in the initial invasion of prostate cancer through the epithelial basal lamina and into the surrounding stroma.     

High-level expression of a novel epitope of CD59 identifies a subset of CD34+ bone marrow cells highly enriched for pluripotent stem cells

To further define the hierarchy of human hematopoietic progenitor cells, we have attempted to identify antibodies to cell-surface molecules expressed on CD34+ progenitor cell subsets. Herein we describe the utility of a new monoclonal antibody, HCC-1, which binds to a novel epitope of CD59 differentially expressed among CD34+ progenitor cells. HCC-1 subdivides the adult marrow CD34+ population into HCC-1high and HCC-1low/- fractions of approximately equal size. Cobblestone area-forming cells (CAFC) in long-term bone marrow culture were enriched 10-30-fold in CD34+HCC-1high cells compared with CD34+HCC1-low/- cells and two-fold compared with CD34+ cells. When injected into fetal human bone fragments implanted in SCID mice, the CD34+HCC-1high population showed potent engrafting activity leading to the production of myeloid, lymphoid, and erythroid elements, as well as the retention of progenitor cell phenotype. These studies demonstrate that the CD34+HCC-1high population contains primitive pluripotent hematopoietic stem cells. No hematopoietic engrafting activity was detected in the CD34+HCC-1low/- population. Consistent with this finding, simultaneous five-color flow cytometric analysis revealed that HCC-1high cells include virtually all CD34+Thy-1+Lin- cells, a cell population previously characterized as highly enriched for primitive pluripotent hematopoietic stem cells. The ability of CD34+ cells divided into subsets by HCC-1 to produce T cells was assessed by transplantation of sorted cells into human fetal thymus implanted into SCID mice. A higher frequency of thymus-engrafting activity was observed in the CD34+HCC-1high than in the CD34+HCC-1low/- population. Consistent with the limited ability to engraft in the SCID-hu thymus model, the CD34+HCC-1low/- population was shown to contain a low frequency of CD34+CD10+ lymphoid progenitor cells. We conclude that the HCC-1 epitope is expressed at high levels on a subset of CD34+ cells that contain virtually all primitive pluripotent hematopoietic stem cells and that the population of CD59 molecules expressed on CD34+ cells is not homogeneous.     

Leptin increases glucose transport and utilization in skeletal muscle in vitro

1. The present study examines the effect of leptin on glucose transport and metabolism in incubated soleus muscle from male lean albino rats. 2. Insulin (100 microU/ml) increased glucose uptake by twofold while the leptin group (100 nmol/l) reached 75% of the insulin response after 1 hr of incubation. However, leptin did not potentiate the insulin effect on glucose uptake in soleus muscle. 3. Leptin elicited a significant increase (27.7%) in total lactate production, accompanied by a three-fold increment in glycogen synthesis from [U-14C]D-glucose. 4. Insulin raised glycogen synthesis by sixfold. The leptin plus insulin group increased glycogen synthesis by eightfold, which is equivalent to the sum of the separated leptin and insulin groups. 5. Leptin per se exerts an insulin-like effect stimulating glucose uptake, glycogen synthesis, and lactate formation and also seems to potentiate the effect of insulin on glucose incorporation into glycogen in incubated soleus muscle.     

Phenotype and genotype of advanced premalignant head and neck lesions after chemopreventive therapy

BACKGROUND: The goal of chemoprevention is to reduce the risk of cancer development by reversing or blocking the tumorigenic process through the use of pharmacologic or natural agents. To determine the potential role of genetic alterations in assessing cancer risk and in evaluating the efficacy of chemopreventive agents, we studied 22 patients with advanced premalignant lesions of the head and neck who were part of a prospective cancer prevention trial that is investigating a regimen of 13-cis-retinoic acid, interferon alfa, and alpha-tocopherol administered for 12 months or until disease progression. METHODS: We used polymerase chain reaction analysis of microsatellite DNA sequences in cells from precancerous lesions to determine the frequencies of genetic alterations--namely, loss of heterozygosity (LOH) and microsatellite instability--at chromosomal loci that are commonly deleted in head and neck cancer. RESULTS: Prior to treatment, 17 (81%) of 21, eight (44%) of 18, and eight (42%) of 19 patients who were informative (i.e., heterozygous) at chromosomes 9p21, 3p14, and 17p13, respectively, exhibited LOH in at least one of their lesion biopsy specimens. Among nine patients who exhibited LOH at chromosome 9p21 in pretreatment biopsy specimens and who had completed at least 5 months of therapy, the genetic loss persisted in eight--including three of the four patients who exhibited complete histologic responses (i.e., no evidence of dysplasia in their biopsy specimens). IMPLICATION: Our data suggest that clinical and histologic assessments of the response to chemopreventive agents may be insufficient to determine their efficacy and that critical genetic alterations could be used as independent biomarkers to augment the ability to evaluate the efficacy of such agents.     

Granulocyte colony-stimulating factor (G-CSF) dose-dependent efficacy in peripheral blood stem cell mobilization in patients who had failed initial mobilization with chemotherapy and G-CSF

For 10 consecutive patients in our unit who did not show a significant rise in blood progenitor cells within 14 days following chemotherapy and G-CSF, we increased the G-CSF dose from 5 to 10 microg/kg/day (n = 9) or from 10 to 15 microg/kg/day (n = 1). As a result, there were significant increases in total yield as well as yield per apheresis of mononuclear cells, CD34+ cells and CFU-GM (P < 0.025, <0.01 and <0.005, respectively). After G-CSF dose escalation, six of the 10 patients had sufficient CD34+ cells for performing transplantation. These results demonstrate a dose-dependent response of progenitor cell mobilization by G-CSF when used in combination with chemotherapy. Moreover, increasing the dose of G-CSF as late as the third week of mobilization may still provide sufficient cell yield even with patients who did not show a significant mobilization with conventional doses of G-CSF.     

Hazard assessment techniques used in the chemical industry and their possible uses elsewhere

The traditional methods used for the identification and assessment of hazards in the chemical industry are based on experience, but increased economic and environmental restrictions on its operations have required the development of other methods to ensure that an acceptable level of safety is maintained. The present paper briefly reviews the traditional methods, describes in mire detail the basis of the newer methods, namely hazard and operability studies and hazard analysis, and considers how they might be more widely used in assessing fire hazards.     

Biotransformation of an antihypertensive arylalkylamine analogue in the rat

1. The excretion and metabolism of N-[2-(3,4-dimethoxyphenyl)ethyl]-5-methoxy-N,alpha-dimethyl-2-(phenyl ethynyl) benzenepropanamine (RWJ-26240) in the Wistar rat has been investigated after a single oral dose of 14C-RWJ-26240 (50 mg/kg free base). 2. Plasma samples were obtained for 24 h after dosing and urine and faecal samples were collected over 8 days, and they accounted for 0.9 and 96% of the dose, respectively. 3. Representative samples of plasma, urine and faecal samples were purified for metabolite isolation and identification using HPLC, tlc, mass spectra (CI and EI), 1H-NMR and derivatization. 4. Unchanged RWJ-26240 plus 11 metabolites were identified and accounted for > 80% of the sample radioactivity. 5. Four metabolic pathways for RWJ-26240 are proposed; namely (1) N-demethylation, (2) O-demethylation, (3) phenyl hydroxylation and (4) N-dealkylation. Pathways 1-3 appeared to be quantitatively more important.     

Physiological response of Lactobacillus plantarum to salt and nonelectrolyte stress

In this report, we compared the effects on the growth of Lactobacillus plantarum of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K+ (or Na+) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.