A novel galactosyl-binding lectin from the plasma of the blood clam,Anadara granosa (L) and a study of its combining site

Control Analysis has been carried out in the first steps of a rat liver glycolytic system. Attention has been focused on the effect of several glucose concentrations on the control, particularly regarding the role of glucokinase. From kinetic studies of the whole metabolic system we have obtained information on the flux variation under different glucose concentrations. This information together with the kinetics of glucokinase has allowed us to calculate Flux Control and Elasticity Coefficients for glucokinase and the Response Coefficient of the system with respect to glucose. The changes in of the value of Flux Control Coefficients demonstrates that in conditions of low glucose concentration, glucokinase is the main enzyme in controlling the flux through the pathway, but at high glucose concentration the control moves to phosphofructokinase. Next, we have compared our results with those obtained with the shortening and titration method, previously described (Torres, N.V., Mateo, F., Mélendez-Hevia, E. and Kacser, H., (1986) Biochem. J. 234, 169–174; Torres, N.V. and Meléndez-Hevia, E. 1991. Molec. Cell. Biochem. 101, 1–10). Furthermore, from knowledge of the enzyme kinetics of the system we have been able to build a model of the pathway that allows us computer similation of its behavior and calculation of the Flux Control Coefficient profile at different glucose concentrations. By the three methods the results correlate, supporting the use of the pathway substrate as external modulator of the metabolic system as a tool for practical application of Control Analysis.     

Selection of Lactobacillus acidophilus strains for use in “acidophilus products”

Recent studies by DNA-DNA hybridization revealed that strains now designated as L. acidophilus, can be divided into several groups and only one group should be classified as L. acidophilus. We studied several phenotypic characteristics in representative strains from the six DNA-homology groups of L. acidophilus. No group specific pattern was observed among the strains for fermentation of eight carbohydrates, growth at 15 and 45°C, resistance to 0.2% oxgall, lysis by lysozyme or sensitivity to 17 antibiotics. However, some differences among groups were observed in -galactosidase (-gal) activity and surface layer (s-layer) protein. Strains in B1 do not have a s-layer or -gal while B2 strains also lack a s-layer but do possess -gal. All strains in groups A1, A2, A3 and A4, capable of growing in lactose, have -gal activity and also have a s-layer composed of protein subunits of different molecular weights (MW). Strains in A1 homology group have a s-layer with 46 Kd protein subunits while strains in other A groups have s-layer protein subunits that varied in MW within each group. On the basis of these two traits several isolates of unknown homology groups have been tentatively placed in A1, B1 or B2 groups. L. acidophilus from A1 group showed strain variation in -gal specific activity and rate of acid production and growth. For use in dietary adjuncts, L. acidophilus strains should be selected for these three and other desirable traits. They should be maintained and grown in media containing lactose.     

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     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

Proton magnetic resonance studies of bradykinin antagonists

Bradykinin (BK) is a peptide hormone with sequence Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹ and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, β-branched D -aliphatic residues at position 7 combined with bulky L -aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D -Arg⁰, Hyp³, Thi⁵, D -Cpg⁷, Cpg⁸]-BK [Cpg: α-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L -proline; Thi: β-(2-thienyl)-L -alanine] ( I ), [D -Arg⁰, Hyp³, D -Cpg⁷, Cpg⁸] -BK ( II ), as well as its variant with D -Cpg⁷ replaced by Cpg⁷, namely [D -Arg⁰, Hyp³, Cpg⁷, Cpg⁸]-BK ( III ). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II , in direct correlation with the peptide activities. No turn-like structure was found for residues 6–9. In peptide III , a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists ( I , II ) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D -Cpg⁷]-BK ( IV ) showed no defined secondary structure. © 1993 John Wiley & Sons, Inc.     

Use of the Reverse Transcription-Polymerase Chain Reaction for the Detection of Pelargonium Flower Break Carmovirus

A polymerase chain reaction (PCR)-based assay was used to detect pelargonium flower break carmovirus (PFBV) in total RNA extractions made from infected Pelargonium plants. Extracts were reverse transcribed (RT) and the resultant cDNA was amplified by PCR, using oligonucleotide primers specific for 343, 510 and 832 base pair fragments of the RNA-dependent RNA polymerase gene of PFBV.
The specificity and sensitivity of RT-PCR were compared with the enzyme-linked immunosorbent assay (ELISA) for the detection of PFBV in Pelargonium tissues. The virus could be detected efficiently in high dilutions of sap from infected plants and at low concentrations of purified virus. Although ELISA is a powerful tool for virus detection, RT-PCR was over 1000 times more sensitive in detecting PFBV in leaf extracts of infected Pelargonium than was ELISA. The limit of detecting PFBV RNA by RT-PCR was 200 fg, compared with 200 pg of virus by ELISA.     

Rat Brain 3-Hydroxy-3-Methylglutaryl-CoA Reductase: Subcellular Distribution and Cold Lability

Abstract: Data are provided indicating that the rat brain 3-hydroxy-3-methyl-glutaryl-CoA reductase is similar to the enzyme from other tissues as far as diurnal rythmicity, cold lability and half-life measurements at 0°C are concerned. The enzyme activity in the brain decreased with age of the animals. Subcellular fractionation studies demonstrate that while 77% of the activity was associated with the microsomal fraction, 19% of the enzyme activity was recovered in the mitochondrial fraction. The possible function of such a mitochondrially located 3-hydroxy-3-methylglutaryl-CoA reductase in rat brain is discussed.     

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5–6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2–3-fold). There were no α-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Some structural features of the d-glucan from the seed of Mirabilis jalapa

The cotyledon of the seed of Mirabilis jalapa was found to contain a d-glucan. Methylation, periodate oxidation, and graded and enzymic hydrolysis studies were conducted to elucidate its structure. For every 38 d-glucosyl residues therein, 34 are (1→4)- and 3 are (1→3)-linked; the d-glucosyl unit at the branch point is linked through O-1, O-2, and O-4. In some places in the chain, there are at least three (1→3)-linked d-glucosyl residues in a sequence. Both α- and β-d-glucosidic linkages are present in the polysaccharide, the former preponderating. The d-glucan gave with iodine a faint blue color that had λ_max 420 nm.     

Emerging Molecular Tools for Engineering Phytomicrobiome

Microbial plant interaction plays a major role in the sustainability of plants. The understanding of phytomicrobiome interactions enables the gene-editing tools for the construction of the microbial consortia. In this interaction, microbes share several common secondary metabolites and terpenoid metabolic pathways with their host plants that ensure a direct connection between the microbiome and associated plant metabolome. In this way, the CRISPR-mediated gene-editing tool provides an attractive approach to accomplish the creation of microbial consortia. On the other hand, the genetic manipulation of the host plant with the help of CRISPR-Cas9 can facilitate the characterization and identification of the genetic determinants. It leads to the enhancement of microbial capacity for more trait improvement. Many plant characteristics like phytovolatilization, phytoextraction, phytodesalination and phytodegradation are targeted by these approaches. Alternatively, chemical communications by PGPB are accomplished by the exchange of different signal molecules. For example, quorum-sensing is the way of the cell to cell communication in bacteria that lead to the detection of metabolites produced by pathogens during adverse conditions and also helpful in devising some tactics towards understanding plant immunity. Along with quorum-sensing, different volatile organic compounds and N-acyl homoserine lactones play a significant role in cell to cell communication by microbe to plant and among the plants respectively. Therefore, it is necessary to get details of all the significant approaches that are useful in exploring cell to cell communications. In this review, we have described gene-editing tools and the cell to cell communication process by quorum-sensing based signaling. These signaling processes via CRISPR- Cas9 mediated gene editing can improve the microbe-plant community in adverse climatic conditions.