A novel, specific interaction involving the Csk SH3 domain and its natural ligand.

C-terminal Src kinase (Csk) takes part in a highly specific, high affinity interaction via its Src homology 3 (SH3) domain with the proline-enriched tyrosine phosphatase PEP in hematopoietic cells. The solution structure of the Csk-SH3 domain in complex with a 25-residue peptide from the Pro/Glu/Ser/Thr-rich (PEST) domain of PEP reveals the basis for this specific peptide recognition motif involving an SH3 domain. Three residues, Ala 40, Thr 42 and Lys 43, in the SH3 domain of Csk specifically recognize two hydrophobic residues, Ile 625 and Val 626, in the proline-rich sequence of the PEST domain of PEP. These two residues are C-terminal to the conventional proline-rich SH3 domain recognition sequence of PEP. This interaction is required in addition to the classic polyproline helix (PPII) recognition by the Csk-SH3 domain for the association between Csk and PEP in vivo. NMR relaxation analysis suggests that Csk-SH3 has different dynamic properties in the various subsites important for peptide recognition.     

A model for continuous enzymatic saccharification of cellulose with simultaneous removal of glucose syrup

Cellulase of Trichoderma viride was concentrated in various molecular cutoff membranes, and flux rates and retention of activity were studied under ultra-filtration conditions. Little or no Cellulase was discharged through the membranes tested. The concentrated (5–8-fold) enzymes were used to saccharify finely ground substrate (Solka Floe) in stirred tank (STR) and membrane reactors (MR). A pressure filtration vessel provided with a membrane for simultaneous removal of low molecular weight products (glucose) from the reacting system (Cellulose-Cellulase) is designated as a membrane reactor. Continuous digestion of dense cellulose suspension in the membrane reactor was achieved. Using PM-30 (Amicon) membrane reasonably high mass flux values (9.7–23.3 gals/ft²—day) were obtained in separating glucose from a digest of 30% cellulose suspension. Abcor membrane (HFA 300) was equally effective and necessitated less care in handling. Nearly 14% glucose concentration has been achieved in less than 50 hrs in STR by digesting a 30% cellulose suspension. Based on experimental data a model system is proposed for the continuous steady state Saccharification of ground substrate in which there is continuous removal of concentrated glucose syrup, and a feedback of enzyme.     

Intravenous tyramine response in migraine before and during treatment with indoramin.

We studied the response of 31 migraine sufferers (20 women, 11 men) to intravenous tyramine (the tyraminedose)pressor response test). Patients were treated either with pacebo tablets or indoramin, and alpha-adrenergic blocking agent, in a double-blind crrossover trial. We found that patients with migraine required significantly less tyramine to increase their cystolic blod pressure by 30 mm Hg when compared with matched controls. Indoramin significantly increased the amount of tyramine needed to raise the systolic blood pressure among migraine suffers and reduced the incidence of posttyramine migraine for m 46% while patients were on placebo tablets to 8% when they were receiveing indoramin. There was no association between tyramine sensitivity and a history of premenstrual or dietary migraine, nor was there a significant difference in the indierenence in the incidence of post-tyramine migrain between men women. We conclude that the intravenous tyramine test may be valuable in assessing migraine suffers who will respond to an alpha-advenergic blocking agent such as indoramin.     

IgA nephropathy in adults: immunohistologic findings and clinical course

Laboratory examination of specimens from 123 consecutive renal biopsies performed at Victoria General Hospital, Halifax revealed six cases of mesangial deposition, predominantly of IgA, unassociated with systemic disorders. Immunohistologic examination showed deposits of only IgA in one specimen, IgA and IgG in two and IgA, IgG and IgM in three. Glomerular deposits of C3 were seen in five of the specimens, and properdin was seen in three. Glomeruli in all the specimens showed increased matrix and increased numbers of cells in the mesangium. Electron microscopy revealed deposits in the mesangium or capillary wall in all five of the specimens so studied. All six patients had proteinuria, four had microscopic hematuria, and three had hypertension; in one patient the disease progressed to renal failure.     

A role for repressive histone methylation in cocaine-induced vulnerability to stress

Substance abuse increases an individual's vulnerability to stress-related illnesses, which is presumably mediated by drug-induced neural adaptations that alter subsequent responses to stress. Here, we identify repressive histone methylation in nucleus accumbens (NAc), an important brain reward region, as a key mechanism linking cocaine exposure to increased stress vulnerability. Repeated cocaine administration prior to subchronic social defeat stress potentiated depressive-like behaviors in mice through decreased levels of histone H3 lysine 9 dimethylation in NAc. Cre-mediated reduction of the histone methyltransferase, G9a, in NAc promoted increased susceptibility to social stress, similar to that observed with repeated cocaine. Conversely, G9a overexpression in NAc after repeated cocaine protected mice from the consequences of subsequent stress. This resilience was mediated, in part, through repression of BDNF-TrkB-CREB signaling, which was induced after repeated cocaine or stress. Identifying such common regulatory mechanisms may aid in the development of new therapies for addiction and depression.     

Antibody directed targeting of methotrexate-containing small unilamellar vesicles

Summary The potential of antibody-linked SUVs containing MTX in anticancer therapy was investigated. The SUVs, mean diameter 50±20 nm, were prepared by probe sonication of MTX-containing MLVs and were covalently linked either to a RAMG or NRG. After incubation with M21 melanoma cells for 2 h, RAMG-linked SUVs showed 2 and 4 times more binding than NRG-linked MTX-containing SUVs or MTX-containing SUVs unlinked to any Ig. Furthermore, on incubating M21 melanoma cells with RAMG-linked ³H MTX-containing SUVs for 2, 4, and 8 h at 4° C or 37° C, a higher radioactivity was associated with cells at 37° C than at 4° C. Membrane immunofluorescence revealed aggregation of and cap formation by RAMG-linked SUVs after 2 h (37° C) and endocytosis at 4 and 8 h at 37° C. Electron microscopic and autoradiographic studies confirmed aggregation of ³H MTX-containing SUVs around and on the surface of M21 cells. Electron microscopy also revealed these SUVs inside invaginations of and under the plasma membrane of melanoma cells. A colony inhibition assay showed that RAMG-linked, MTX-containing SUVs were 60 times, 8 times, and 4.5 times more growth inhibitory than free MTX, NRG-linked MTX-containing SUV, and MTX-containing SUVs unlinked to any Ig, but not toxic to a human kidney cancer line (that did not react with RAMG). Abbreviations used: DPPC, DL- -dipalmitoyl phosphatidylcholine; DTT, dithiothreitol; MTX, methotrexate; (MTX)SUV or MLV, MTX-containing SUV or MLV; MLV, multilamellar vesicle; NRG, normal rabbit immunoglobulin G; RAMG, rabbit antimelanoma IgG; SA, stearylamine; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate; SUV, small unilamellar vesicle; CHOL, cholesterol; LUV, large unilamellar vesicle; Ig, immunoglobulin; PDP-SA, N-[3-(2-pyridyldithio)-propinyl]stearylamine