Growth and dissemination of a newly-established murine B-cell lymphoma cell line is inhibited by multimeric YIGSR peptide

B-cell lymphoma frequently shows simultaneous dissemination to multiple organs. It also occasionally involves bone and causes osteolytic lesions. To study the mechanisms responsible for this capacity of lymphoma cells to grow in different tissue microenvironments and search for effective therapeutic interventions for this hematological malignancy, we established a new murine B-cell lymphoma cell line named MH-95. The tumor disseminated to multiple organs including the lung, liver, kidney, spleen and lymph nodes within 2 weeks after subcutaneous inoculation in nude mice. In addition, the tumor also grew in bone and caused osteoclastic osteolytic lesions. Thus, this tumor model mimics the behavior in many ways of B-cell lymphoma in humans. We studied the role of laminin, a major component of the basement membrane, in this model, since although it has been implicated in solid tumor metastasis, little is known about the involvement of laminin in the growth of B-cell lymphoma in bone and other organs. Immunohistochemical examination showed strong laminin expression in the stroma of the primary subcutaneous tumor and tumors in the bone and other organs. Systemic administration of the antagonistic laminin peptide YIGSR decreased primary tumor growth and tumor cell deposit in the bone, liver and kidney. In addition, the peptide also decreased apparent neovascularization in the tumor, suggesting that the peptide suppressed angiogenesis presumably due to inhibition of laminin binding to its receptors. These results demonstrate that the MH-95 B-cell lymphoma cells express laminin and suggest that laminin plays a critical role in the growth and simultaneous dissemination of tumor cells to multiple organs, similar to what has been described in solid tumors. The results also suggest that suppression of angiogenesis through interfering with laminin actions may be a useful adjuvant therapy for B-cell lymphoma.     

Environmental influence on immune inhibition of release of herpes simplex virus from cells

Immune inhibition of virus release (IVR) of herpes simplex virus type 1 (HSV-1) from baby hamster kidney cells (BHK-21) was mediated by antisera against BHK cells, HSV-1, human fibronectin and mouse heparan sulphate proteoglycan and was irreversible for at least 24 h following removal of antiserum. Enhancement of IVR by calf serum depletion of growth media was obtained in varying measure using each of these antisera and also by treatment of virus-infected cells by the lectin concanavalin A. Enhancement was reversible by replenishment of growth media with bovine serum components larger than 12 kD but this only occurred when replenishment was instituted prior to virus infection. There was also reversibility to varying degree following replenishment by ovine, equine and human serum which indicates that this phenomenon is not species specific. In addition to the presence of relevant antigens on the cell surface, IVR may also require an alteration in the cell membrane; this is evidenced by the absence of anticellular serum-mediated IVR when treatment was introduced less than 6 h after virus infection, suggesting that a certain level of alteration or possibly cell damage - in this case virus induced - is necessary. Enhancement of IVR by calf serum depletion would seem to operate through a specific alteration in the virus-infected cell membrane as serum-depleted cells did not show histological alteration and were able to replicate HSV-1 to usual titres; it is possible that this enhancement may represent an as yet unidentified host defence mechanism whereby extracellular release of virus will be reduced in ischaemic or necrotic tissue in the course of infectious inflammatory processes.     

BDNF overexpression induces differential increases among subsets of sympathetic innervation in murine back skin

Besides their recognized dependence on nerve growth factor (NGF) during development, the dependence of mature sympathetic ganglion neurons on other neurotrophins is still unclear. Here, we have investigated the sympathetic innervation of back skin in mice overexpressing brain-derived neurotrophic factor (BDNF) under the alpha-myosin heavy-chain promoter, as well as in BDNF knockout (-/-) mice. Compared with wild-type controls, the dorsal skin of BDNF overexpressing mice displayed a significantly enhanced number of adrenergic, tyrosine hydroxylase-immunoreactive (IR) nerve fibres, while cholinergic or peptidergic sensory nerve fibres appeared unaltered. The adrenergic hyperinnervation in dorsal skin of BDNF overexpressing mice was most pronounced in the arrector pili muscle of hair follicles, while no increase of tyrosine hydroxylase-or neuropeptide Y-IR fibres associated with subcutaneous blood vessels was found. Instead, back skin of BDNF knockout (-/-) mice contained significantly fewer tyrosine hydroxylase-IR dermal nerve fibres than wild-type animals. This suggests that BDNF plays an important role in the control of different subsets of adrenergic innervation in murine back skin, and indicates that paravertebral sympathetic ganglia display a previously unrecognized differential BDNF-dependence in vivo.     

Disruption of c-Fos leads to increased expression of NAD(P)H:quinone oxidoreductase1 and glutathione S-transferase

Regulation of the basal and induced expression of detoxifying enzymes such as NAD(P)H:quinone oxidoreductasel (NQO1) and glutathione S-transferase (GST) by the antioxidant response element (ARE) is important for cellular protection against oxidative stress. The ARE contains AP1 and AP1-like elements and is known to bind to several leucine zipper proteins including c-Fos. Previous studies (Venugopal, R., and Jaiswal, A.K. (1996) Proc. NatL Acad. Sci. USA 93, 14960-14965) have shown that overexpression of c-Fos in transfected cells leads to repression of ARE-mediated gene expression. In the present report, we used c-Fos-/- mice and investigated the physiological (in vivo) role of c-Fos in repression of the NQO1 and GST genes expression. The analysis of enzyme activity levels showed significant increases in NQO1 and GST activities in several tissues of c-Fos-/- mice, as compared with wild type (c-Fos+/+) mice. The increases in enzyme activities were supported by Wetern analysis of respective proteins. Western analyses showed significant increases in the expression of NQO1 in kidney, liver and skin tissues of c-Fos-/- mice, as compared with wild type (c-Fos+/+) controls. Western analyses also demonstrated an increased expression of the GST Ya gene in kidney and liver tissues of the c-Fos-/-mice. These results confirm a negative (repressive) role for c-Fos in the expression of NQO1, GST Ya, and other detoxifying enzyme genes.     

Identification of the quinone cofactor in mammalian semicarbazide-sensitive amine oxidase

Mammalian semicarbazide-sensitive amine oxidase (SSAO) enzymes have been classified as EC 1.4.3.6 [amine:oxygen oxidoreductase (deaminating)(copper-containing)]. However, both the identity of the quinone cofactor and the presence of copper remain unconfirmed, and SSAO has proved impossible to purify to homogeneity in sufficient yield to permit cofactor identification. To circumvent this problem, we have partially purified SSAO enzymes from bovine and porcine aortae and have established, with a redox-cycling assay, that no other quinoproteins were present in enzyme preparations. Enzymes were then derivatized with (p-nitrophenyl)hydrazine (p-NPH), which forms a covalent yellow complex with the quinone cofactor. Visible absorbance spectra of derivatized bovine and porcine enzymes (respective lambdamax values 456 and 476 nm at neutral pH, shifting to 580 and 584 nm in 2 M KOH) were consistent with the presence of (2,4,5-trihydroxyphenyl)alanine quinone (TPQ) as cofactor. Resonance Raman spectra were essentially identical to that for pea seedling amine oxidase, a known TPQ-containing enzyme. Extensive digestion of SSAO enzymes, and of porcine kidney diamine oxidase, with pronase E yielded species with identical chromophoric properties characteristic of the dipeptide, TPQ(p-NPH)-Asp. Thermolytic digestion of porcine SSAO gave two cofactor-containing peptides that contained a TPQ consensus sequence, Asn-X-Asp-Tyr-Tyr, where X is a blank cycle corresponding to TPQ. N-terminal sequencing of whole enzymes revealed a membrane-spanning region typical of an extracellular type II glycoprotein. These results confirm the presence of TPQ in mammalian membrane-bound SSAO ectoenzymes.     

In vivo model of adeno-associated virus vector persistence and rescue

Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206-212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue.     

Population analysis of the pharmacokinetics of tiagabine in patients with epilepsy

PURPOSE: In two open-label long-term safety studies, we determined tiagabine (TGB) pharmacokinetics in patients with epilepsy. METHODS: In all, 2,147 plasma samples from 511 patients who participated in the studies were available. The total daily dose ranged from 2 mg administered once daily to 80 mg administered in four doses. A one-compartment model with first-order absorption and elimination was used to fit the TGB plasma concentration-time data, with a population pharmacokinetic approach. RESULTS: The patients' average (+/-SD) weight and age were 73.8+/-20.7 kg and 32.1+/-12.3 years. The most significantly factor affecting TGB pharmacokinetics was concomitant administration of other antiepileptic drugs (AEDs). The central clearance value in patients receiving AEDs known to induce hepatic drug metabolism was 21.4 L/h, a value 67% higher than the central clearance estimate obtained for the patients receiving AEDs not known to affect hepatic drug metabolism (12.8 L/h). There was no evidence of any dose or time effect, indicating that TGB pharmacokinetics are linear. TGB pharmacokinetics were not different in white, black, or Hispanic patients, although our ability to explore racial effects was limited since 90% of the patients were white. No other demographic variables (including age and smoking) or any clinical chemistry measurements (including bilirubin, SGOT, and SGPT) were important in explaining the variability in the clearance estimates. CONCLUSIONS: TGB pharmacokinetics are linear, influenced by enzyme-inducing AEDs, and largely unaffected by other demographic variables.     

Synthesis and characterization of bay region halohydrins derived from Benzo[a]pyrene diol epoxide and their role as intermediates in halide-catalyzed cis adduct formation

The bay region epoxide of benzo[a]pyrene (anti-BPDE) alkylates DNA to form adducts with >98% trans stereochemistry. Halide ions catalyze this reaction; however, this pathway is characterized by the formation of adducts with altered cis stereochemistry. Bay region halohydrins are possible intermediates in these reactions, but are too unstable to be isolated from aqueous solutions. However, we successfully synthesized halohydrins in tetrahydrofuran (THF) by treatment of anti-BPDE with the corresponding lithium halide salt in the presence of acetic acid. Absorbance and CD spectroscopy clearly indicated the formation of chloro-, bromo-, and iodohydrins. The structure and stereochemistry of the chlorohydrin was established by NMR. Chloride addition is exclusively at the benzylic position of the epoxide, and the stereochemistry of the C-9 and -10 positions is trans. The long-wavelength absorbance band in the chloro-, bromo-, and iodohydrin is red-shifted 7, 13, and 22 nm, respectively, relative to the hydrolysis product of anti-BPDE. The ellipticity of the same absorbance band in CD spectra of enantiomerically pure halohydrins is opposite in sign compared to that of the corresponding anti-BPDE enantiomer. The relative stability of these derivatives is chlorohydrin > bromohydrin > iodohydrin. The chloro- and bromohydrins were isolated, but the iodohydrin decomposed during this manipulation. The addition of 500 mM chloride decreased the hydrolysis rate of the chlorohydrin 4-fold in 50% THF/buffer. Direct evidence for the transient formation of the iodohydrin in aqueous buffer/acetone mixtures was obtained by absorbance spectroscopy. At 1 M chloride, bromide, and iodide, alkylation of deoxyadenosine by anti-BPDE in aqueous buffer yields 85, 91, and 92% cis adducts, respectively. In the absence of halide, alkylation of deoxyadenosine in buffer by anti-BPDE, the chlorohydrin, and the bromohydrin yields 32, 65, and 83% cis adducts, respectively.     

Glycemic index--a new tool in sport nutrition?

The glycemic index (GI) provides a way to rank foods rich in carbohydrate (CHO) according to the glucose response following their intake. Consumption of low-GI CHO foods may attenuate the insulin-mediated metabolic disturbances associated with CHO intake in the hours prior to exercise, better maintaining CHO availability. However, there is insufficient evidence that athletes who consume a low-GI CHO-rich meal prior to a prolonged event will gain clear performance benefits. The ingestion of CHO during prolonged exercise promotes CHO availability and enhances endurance and performance, and athletes usually chose CHO-rich foods and drinks of moderate to high GI to achieve this goal. Moderate- and high-GI CHO choices appear to enhance glycogen storage after exercise compared with low GI CHO-rich foods. However, the reason for this is not clear. A number of attributes of CHO-rich foods may be of value to the athlete including the nutritional value of the food or practical issues such as palatability, portability, cost gastric comfort, or ease of preparation.