Hepatic arterial spin labelling MRI: an initial evaluation in mice.

The development of strategies to combat hepatic disease and augment tissue regeneration has created a need for methods to assess regional liver function. Liver perfusion imaging has the potential to fulfil this need, across a range of hepatic diseases, alongside the assessment of therapeutic response. In this study, the feasibility of hepatic arterial spin labelling (HASL) was assessed for the first time in mice at 9.4 T, its variability and repeatability were evaluated, and it was applied to a model of colorectal liver metastasis. Data were acquired using flow-sensitive alternating inversion recovery-arterial spin labelling (FAIR-ASL) with a Look-Locker readout, and analysed using retrospective respiratory gating and a T1 -based quantification. This study shows that preclinical HASL is feasible and exhibits good repeatability and reproducibility. Mean estimated liver perfusion was 2.2 ± 0.8 mL/g/min (mean ± standard error, n = 10), which agrees well with previous measurements using invasive approaches. Estimates of the variation gave a within-session coefficient of variation (CVWS) of 7%, a between-session coefficient of variation (CVBS) of 9% and a between-animal coefficient of variation (CVA) of 15%. The within-session Bland-Altman repeatability coefficient (RCWS) was 18% and the between-session repeatability coefficient (RCBS) was 29%. Finally, the HASL method was applied to a mouse model of liver metastasis, in which significantly lower mean perfusion (1.1 ± 0.5 mL/g/min, n = 6) was measured within the tumours, as seen by fluorescence histology. These data indicate that precise and accurate liver perfusion estimates can be achieved using ASL techniques, and provide a platform for future studies investigating hepatic perfusion in mouse models of disease.

Association between vitamin D and diabetic neuropathy in a nationally representative sample: results from 2001-2004 NHANES.

AIMS: To evaluate the association between vitamin D insufficiency and peripheral neuropathy in a nationally representative sample of adults with diagnosed diabetes. METHODS: Vitamin D concentrations, medical examination variables and questionnaire results from the 2001-2004 National Health and Nutrition Examination Survey were analysed for adults ≥ 40 years old with diagnosed diabetes (unweighted n = 591, weighted n = 8.82 million). Neuropathy was defined as self report of peripheral neuropathy symptoms of painful sensation, tingling, numbness or loss of feeling in hands or feet. Additionally, Semmes-Weinstein monofilament test results were used as an indicator of neuropathy. Insufficient vitamin D was characterized as < 30 ng/ml. RESULTS: In the weighted population, 81% of adults with diabetes had vitamin D insufficiency. Vitamin D insufficiency was more common among Hispanics (92%) and non-Hispanic black people (98%) than among non-Hispanic white people (76%). Within the 3 months preceding the questionnaire, 50% reported experiencing pain or numbness (paresthesia) in their hands or feet; 37% reported pain or tingling in hands or feet; and 38% reported numbness or loss of feeling in hands or feet. Eight per cent had 4-6 insensate areas on their feet as determined by the Semmes-Weinstein monofilament test. Logistic regressions demonstrate vitamin D insufficiency is associated with the adjusted composite paresthesia measure (odds ratio 2.12; 95% CI 1.17-3.85) and the adjusted numbness measure (odds ratio 2.04; 95% CI 1.18-3.52). CONCLUSIONS: Vitamin D insufficiency is associated with self-reported peripheral neuropathy symptoms even after adjusting for demographic factors, obesity, co-morbidities, use of medications for neuropathy and diabetes duration and control.

Bifunctional DNA alkylator 1,3-bis(2-chloroethyl)-1-nitrosourea activates the ATR-Chk1 pathway independently of the mismatch repair pathway.

The presence of DNA damage initiates signaling through the ataxia-telangiectasia mutated kinase (ATM) and the ATM- and the Rad3-related kinase (ATR), which phosphorylate, thus activating, the checkpoint kinases (Chk) 1 and 2, which leads to cell cycle arrest. The bifunctional DNA alkylator 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is cytotoxic primarily by inducing DNA monoadducts and ultimately, interstrand cross-links, which block DNA replication. In this study, we investigated the activation of the ATR-Chk1 pathway in response to BCNU treatment and the dependence of this response on the DNA mismatch repair (MMR) capacity. Medulloblastoma cells were exposed to low and moderate doses of BCNU, and the effects on this DNA damage signaling pathway were examined. In response to BCNU, Chk1 was found to be phosphorylated at serine 345 and exhibited increased kinase activity. Caffeine and wortmannin, which are broad-spectrum inhibitors of ATM and ATR, reduced this phosphorylation. Cell cycle analysis further revealed an accumulation of cells in the S phase in response to BCNU, an effect that was attenuated by caffeine. Small interfering RNA knockdown of ATR also reduced Chk1 phosphorylation after exposure to BCNU. However, knockdown of ATM had no effect on the observed Chk1 phosphorylation, suggesting that ATR was primarily responsible for Chk1 activation. Analysis of Chk1 activation in cells deficient in MMR proteins MutLalpha or MutSalpha indicated that the DNA damage response induced by BCNU was independent of the MMR apparatus. This MMR-independent activation seems to be the result of DNA interstrand cross-link formation.

Unusual enhancer function in yeast rRNA transcription.

The rRNA genes in most eucaryotic organisms are present in a tandem array. There is substantial evidence that transcription of one of these genes may not be independent of transcription of others. In particular, in the yeast Saccharomyces cerevisiae, the enhancer of rRNA transcription that lies 2.2 kilobases 5' of the transcription initiation site is at least partly within the upstream transcription unit. To ask more directly about the relationship of the tandemness of these genes to their transcription, we have constructed a minirepeat containing two identifiable test genes, with or without enhancer(s). On integration into the URA3 locus, these genes were transcribed by RNA polymerase I. A single enhancer effectively stimulated transcription of both genes by 10- to 30-fold, even when it was located upstream of both or downstream of both. Two enhancers had roughly additive effects. These results suggest a model of enhancer function in tandemly repeated genes.

Phosphorylation of the Saccharomyces cerevisiae equivalent of ribosomal protein S6 has no detectable effect on growth.

The phosphorylation of mammalian ribosomal protein S6 is affected by a variety of agents, including growth factors and tumor promoters, as well as by expressed oncogenes. Its potential role in the regulation of protein synthesis has been the object of much study. We have developed strains of Saccharomyces cerevisiae in which the phosphorylatable serines of the equivalent ribosomal protein (S10) were converted to alanines by site-directed mutagenesis. The S10 of such cells is not phosphorylated. Comparison of these cells with the parental cells, whose genomes differ by only six nucleotides, revealed no differences in the lag phase or logarithmic phase of a growth cycle, in growth on different carbon sources, in sporulation, or in sensitivity to heat shock. We conclude that in S. cerevisiae the phosphorylation of ribosomal protein S10 may play no role in regulating the synthesis of proteins. This conclusion leads one to ask whether certain protein phosphorylations are simply the adventitious, if easily observable, result of the imperfect specificity of one or another protein kinase.

The rRNA enhancer regulates rRNA transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the rRNA genes are organized as a tandem array of head-to-tail repeats. An enhancer of rRNA transcription is present just at the end of each transcription unit, 2 kb away from the next one. This enhancer is unusual for S. cerevisiae in that it acts both upstream and downstream of, and even across, genes. The role of the enhancer in the nutritional regulation of rRNA transcription was studied by introducing a centromere plasmid carrying two rRNA minigenes in tandem, flanking a single enhancer, into cells. Analysis of the transcripts from the two minigenes showed that the enhancer was absolutely required for the stimulation of transcription of rRNA that occurs when cells are shifted from a poor carbon source to a good carbon source. While full enhancer function is provided by a 45-bp region at the 3' end of the 190-bp enhancer, some activity was also conferred by other elements, including both a T-rich stretch and a region containing the binding sites for the proteins Reb1p and Abf1p. We conclude that the enhancer is composed of redundant elements and that it is a major element in the regulation of rRNA transcription.

Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft.

A methylator-resistant human glioblastoma multiforme xenograft, D-245 MG (PR), in athymic nude mice was established by serially treating the parent xenograft D-245 MG with procarbazine. D-245 MG xenografts were sensitive to procarbazine, temozolomide, N-methyl-N-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, 9-aminocamptothecin, topotecan, CPT-11, cyclophosphamide, and busulfan. D-245 MG (PR) xenografts were resistant to procarbazine, temozolomide, N-methyl-N-nitrosourea, and busulfan, but they were sensitive to the other agents. Both D-245 MG and D-245 MG (PR) xenografts displayed no O6-alkylguanine-DNA alkyltransferase activity, and their levels of glutathione and glutathione-S-transferase were similar. D-245 MG xenografts expressed the human mismatch repair proteins hMSH2 and hMLH1, whereas D-245 MG (PR) expressed hMLH1 but not hMSH2.

Do infants have the horizontal bias?

A robust set of studies show that adults make more horizontal than vertical and oblique saccades, while scanning real-world scenes. In this paper we study the horizontal bias in infants. The directions of eye movements were calculated for 41 infants (M=8.40 months, SD=3.74, range=3.48-15.47) and 47 adults (M=21.74 years, SD=4.54, range=17.89-39.84) while viewing 28 real-world scenes. Saccade directions were binned to study the proportion of saccades in the horizontal, vertical and oblique directions. In addition, saccade directions were also modeled using a mixture of Von Mises distributions, to account for the relatively large amount of variance in infants data. Horizontal bias was replicated in adults and also found in infants, using both the binning and Von Mises approach. Moreover, a developmental pattern was observed in which older infants are more precise in targeting their saccades than younger infants. That infants have a horizontal bias is important in understanding infants' eye movements. Future studies should account for the horizontal bias in their designs and analyses.

Schedule-dependent activity of temozolomide plus CPT-11 against a human central nervous system tumor-derived xenograft.

Temozolomide, an imidazole tetrazinone, and CPT-11, a camptothecin derivative, have previously been shown to have anti-central nervous system tumor activity in laboratory and clinical studies. The current experiments were designed to evaluate the activity of temozolomide plus CPT-11 against a malignant glioma-derived xenograft, D-54 MG, growing s.c. in athymic nude mice. The initial schedule of i.p. drug administration was temozolomide at 0.1 LD10 on day 1 and CPT-11 at 0.1 LD10 on days 1-5 and 8-14. The combination of these two agents produced greater than additive activity against D-54 MG. This enhanced activity was maintained when the initial administration of CPT-11 was delayed to day 3 or day 5. However, when CPT-11 was administered first on day 1 using 0.5 LD10 (for the single dose schedule) followed by temozolomide (0.1 LD10) 5 h, 3 days, or 5 days later, the enhancement of activity was substantially reduced. These results demonstrate that the combination of temozolomide plus CPT-11 displays a schedule-dependent enhancement of antitumor activity, suggest a mechanistic explanation for the enhanced activity, and provide the rationale for a Phase I trial of this regimen.

Analysis of DNA mismatch repair proteins in human medulloblastoma.

During replication, the primary function of the eukaryotic DNA mismatch repair (MMR) system is to recognize and correct mismatched base pairs within the DNA helix. Deficiencies in MMR have been reported previously in cases of hereditary nonpolyposis colorectal cancer and sporadic tumors occurring in a variety of tissues including gliomas. Furthermore, recent evidence indicates that the MMR system may be involved in mediating therapeutic sensitivity to alkylating agents. In this study, 22 neoplastic tissue samples from 22 patients who underwent surgical resection for medulloblastoma, a common cerebellar tumor of childhood, were assayed for the presence or absence of MMR polypeptides using Western blot and immunohistochemical techniques. Results from these experiments indicate that the MMR system is not commonly deficient in medulloblastoma.

Effects of oxidized LDL on mononuclear phagocytes: inhibition of induction of four inflammatory cytokine gene RNAs, release of NO, and cytolysis of tumor cells.

A critical step in development of atherosclerosis is the interaction of oxidized low-density lipoprotein (LDL) with mononuclear phagocytes. Oxidized LDL, as well as acetyl-LDL, is rapidly taken up into macrophages via a family of scavenger receptors. We report that macrophages treated with oxidized LDL have markedly lower levels of mRNA specific for the genes MCP-1, TNF-alpha, IL-1 alpha, and KC as measured by Northern blot analyses of lipopolysaccharide (LPS)-stimulated macrophages. By contrast, acetyl-LDL does not inhibit these genes at the doses at which oxidized-LDL is effective. Similar effects are observed whether the LDL is oxidized in the presence of Cu2+ or of Fe2+. Such inhibition also occurs when maleylated bovine serum albumin (BSA), which also clears by one or more scavenger receptors on macrophages, is used as the stimulant. Fe2+ or Cu2+ oxidized LDL inhibits release of nitric oxide when triggered by LPS and direct cytolysis of tumor cells when triggered by maleylated BSA or LPS. Taken together, the data presented indicate that oxidized LDL inhibits induction of several important gene RNAs as well as functional markers that characterize the development of inflammatory and fully activated macrophages.

Myocardial renin-angiotensin extraction in man.

Renin activity, renin substrate, and angiotensin II concentration were measured in the aortic and coronary sinus blood of 14 patients. There was a significant gradient for angiotensin II only. The gradient was diminished in areas of ischaemic myocardium. The study suggests that angiotensin II is partially bound, extracted, or destroyed in the coronary circulation of man.

Transjugular portosystemic shunts in pediatric patients awaiting liver transplantation.

Three pediatric patients from 6 to 11 years of age awaiting liver transplantation for end stage liver disease underwent transjugular intrahepatic portosystemic shunt (TIPS) placement for control of variceal bleeding. Two of the three procedures were performed emergently after endoscopic sclerotherapy failed to stop active bleeding. One procedure was performed electively after multiple prior bleeding episodes. The shunts were created from the middle or left hepatic vein to the left portal vein, and none of the subsequent transplant surgeries was complicated by the presence of the stents. No major or minor complications were related to TIPS placement. Two patients underwent concomitant variceal embolization. Bleeding was successfully controlled in each patient. We conclude that TIPS placement in children is technically feasible, does not complicate subsequent surgery, and is useful treating acute variceal hemorrhage in pediatric patients awaiting liver transplantation.

Synchronous change and perception of object unity: evidence from adults and infants.

Adults and infants display a robust ability to perceive the unity of a center-occluded object when the visible ends of the object undergo common motion (e.g. Kellman, P.J., Spelke, E.S., 1983. Perception of partly occluded objects in infancy. Cognitive Psychology 15, 483-524). Ecologically oriented accounts of this ability focus on the primary of motion in the perception of segregated objects, but Gestalt theory suggests a broader possibility: observers may perceive object unity by detecting patterns of synchronous change, of which common motion is a special case. We investigated this possibility with observations of adults and 4-month-old infants. Participants viewed a center-occluded object whose visible surfaces were either misaligned or aligned, stationary or moving, and unchanging or synchronously changing in color or brightness in various temporal patterns (e.g. flashing). Both alignment and common motion contributed to adults' perception of object unity, but synchronous color changes did not. For infants, motion was an important determinant of object unity, but other synchronous changes and edge alignment were not. When a stationary object with aligned edges underwent synchronous changes in color or brightness, infants showed high levels of attention to the object, but their perception of its unity appeared to be indeterminate. An inherent preference for fast over slow flash rates, and a novelty preference elicited by a change in rate, both indicated that infants detected the synchronous changes, although they failed to use them as information for object unity. These findings favor ecologically oriented accounts of object perception in which surface motion plays a privileged role.

Schedule-dependent activity of irinotecan plus BCNU against malignant glioma xenografts.

PURPOSE: To further evaluate the activity of irinotecan (CPT-11) plus 1,3-bis-(chloroethyl)-1-nitrosourea (BCNU) in the treatment of central nervous system tumor-derived xenografts in athymic nude mice. METHODS: We report studies evaluating the schedule-dependence of this regimen in the treatment of the malignant glioma xenograft D-54 MG. RESULTS: The combination of BCNU and CPT-11 showed the highest enhancement index (2.0-3.3) when BCNU was given on day 1 and CPT-11 was given on days 1-5 and 8-12. Delay of CPT-11 administration to day 3 or day 5 substantially decreased activity with enhancement indices of 1.6-1.8 and 0.6-1.0, respectively. Delay of BCNU administration to day 8 also reduced the CPT-11 activity with enhancement indices of 1.2-1.4. CONCLUSIONS: These results suggest that the presence of a BCNU-induced adduct or possibly crosslink prior to administration of CPT-11 is critical for enhanced activity. Although the mechanism of this enhancement is not currently known, a phase I trial of CPT-11 plus BCNU for adults with recurrent malignant glioma based on these results is in progress.

Multiple DNA repair mechanisms and alkylator resistance in the human medulloblastoma cell line D-283 Med (4-HCR).

PURPOSE: We have previously reported preferential repair of DNA interstrand crosslinks in the 4-hydroperoxycyclophosphamide-resistant human medulloblastoma cell line D-283 Med (4-HCR). We now report further studies that explored the potential mechanisms underlying this repair. METHODS: Limiting dilution assays and Western, Southern, and Northern blots were used to compare specific differences between D-283 Med (4-HCR) and its parental line D-283 Med. RESULTS: D-283 Med (4-HCR) was cross-resistant to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with O6-alkylguanine-DNA alkyltransferase (AGT) levels of 466+/-164 fmol/mg protein; AGT levels in the parental line, D-283 Med, were 76+/-96 fmol/mg. The increase in AGT activity was not a result of gene amplification. Depleting AGT with O6-benzylguanine partially restored sensitivity to BCNU. Both cell lines were deficient in the human mismatch protein MutLalpha. ERCC4 mRNA and poly(ADP-ribose) polymerase levels were similar in both cell lines, and ERCC1 mRNA levels were 2- to 2.5-fold lower in D-283 Med (4-HCR). Topoisomerase I levels were 2- to 2.5-fold higher in D-283 Med compared with D-283 Med (4-HCR). CONCLUSION: These results, while illustrating the multiple differences between D-283 Med and D-283 Med (4-HCR), do not explain the enhanced DNA interstrand crosslink repair seen in D-283 Med (4-HCR).

Modulation of cyclophosphamide activity by O6-alkylguanine-DNA alkyltransferase.

PURPOSE: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)- -nitrosourea, but partial sensitivity is restored after elevated levels of O6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O6-benzylguanine (O6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O6-BG. METHODS: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O6-BG. RESULTS: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. CONCLUSIONS: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.

Biochemical and morphological studies of rat submandibular gland: II. Partial purification of proteins from granule-rich fraction.

Soluble proteins derived from a centrifuged and filtered granule-rich fraction of homogenized rat submandibular gland were analyzed by gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis. Both the granule-rich fraction and final supernatant fraction contained alkaline esterase activity. The major protein component, derived from granules of the convoluted tubules, was further resolved into a series of peptides ranging in molecular weight from 9,000 to 55,000 daltons.

Preoperative embolization of anastomoses of the jugular bulb: an adjuvant in jugular foramen surgery.

We describe the technique of preoperative embolization of the inferior petrosal sinus/anterior condylar vein complex and the posterior condylar vein in three patients undergoing skull base surgery that required opening of the jugular bulb. Contrary to the usual situation, essentially no blood was lost during the operation, resulting in decreased surgical time and reduced risk to the lower cranial nerves.

A large-scale, short-term, media-based weight loss program.

BACKGROUND: This article describes the evaluation of a media-based weight loss and nutrition program. METHODS: Fifteen broadcasts were aired on a Chicago television news program over a three-week period in November of 1986. Some participants (n = 37) received the television program and an accompanying manual, and some (n = 37) received, in addition to the television and media interventions, encouragement to attend self-help groups dealing with obesity. RESULTS: Repeated measures analysis of variance tests were performed, and planned comparisons were conducted only if main effects were significant. At posttesting, those participants attending the self-help groups lost an average of more than nine pounds, whereas those provided only the television program and manual had decreased by less than a pound. Those attending the groups had significantly decreased their percent of dietary fat intake, significantly increased aerobic exercise, and had significantly more hopefulness, motivation, and stimulus control. DISCUSSION: The findings suggest that short-term mass media programs by themselves were probably not very effective, but when supplemented by a self-help manual and support groups may be able to produce significant short-term weight loss.