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M L Scott

Publications and source records attributed to M L Scott.

At least 55 records · Page 3Linked to original sources
PubMed1995

Functional neural regeneration in the feeding system of Aplysia: behavioral recovery correlated with changes in buccal motor output.

1. We tested for functional neural regeneration in the feeding system of Aplysia after bilateral transections or crushes of the cerebral-buccal connectives (CBCs) with the use of behavioral analyses and electrophysiological recordings. 2. Both types of lesion selectively abolished rhythmic consummatory behavior, dramatically increasing bite latency and interbite interval, and decreasing bite magnitude. Appetitive feeding behavior was not affected. 3. About 2 wk after CBC crush, bite latency, bite magnitude, and interbite interval began to recover, as rhythmic biting reappeared; complete recovery of rhythmic biting occurred within 60 days. Rhythmic biting never recovered after transection of the CBCs. 4. The recovery of rhythmic biting was correlated with changes in buccal motor output, which were assessed with the use of in vivo recordings from buccal nerve 4 in freely moving Aplysia. Initially, some bursting in nerve 4 occurred without overt bites; with full recovery of biting, a 1:1 correspondence between bursts in nerve 4 and overt bites returned. 5. CBC lesions caused a functional separation between biting and swallowing; at early times postlesion, subjects displayed apparently normal rhythmic swallowing even though rhythmic biting had been eliminated. However, there was a disruption of the 1:1 correspondence between nerve 4 bursts and swallows, which persisted until consummatory feeding fully recovered. 6. Transection of the CBCs in animals that had fully recovered from a previous CBC crush again abolished rhythmic biting, suggesting that the recovery of consummatory feeding behavior was due to functional neural regeneration of cerebral-buccal connections.

Animals↗
PubMed1994

Characterization of human blood group scFv antibodies derived from a V gene phage-display library.

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

ABO Blood-Group System↗
PubMed1994

Review of the problems involved in using enzymes in blood group serology--provision of freeze-dried ICSH/ISBT protease enzyme and anti-D reference standards. International Council for Standardization in Haematology. International Society of Blood Transfusion.

Proteolytic enzyme preparations and techniques used routinely in blood group serology for the detection of atypical patient antibodies prior to transfusion vary widely and are often poorly standardised. Recent advances have been made in the use of biochemical methods to standardise and stabilise the potency of the enzyme preparations used. A joint working party of the International Council for Standardization in Haematology (ICSH) and the International Society of Blood Transfusion (ISBT) has investigated possibilities for the provision of standards for the protease preparations and techniques. The specification for these standards was that the performance of enzyme reference preparation in the reference technique should be of equivalent sensitivity to the ICSH/ISBT LISS spin indirect antiglobulin test using a titration series of a reference weak anti-D, and be free from false-positive reactions. The working party circulated materials for evaluation in inter-laboratory trials, followed by a laboratory workshop meeting to achieve agreement on the specification for reference materials and methods. Reference freeze-dried papain at 0.6 azoalbumin units and weak anti-D preparations (91/562) have been prepared and validated to meet these specifications. The performance of a test enzyme preparation in the technique for which it is recommended for use should be at least equal to that of the reference papain preparation, by the reference two-stage technique in terms of sensitivity, using a titration series of the reference anti-D, and freedom from false-positive reactions, using six fresh inert sera. The reference papain and weak anti-D can also be used to calibrate the level of proteolytic activity required in other procedures in blood group serology, such as new technology methods for antibody detection, and automated and microplate cell grouping procedures. These preparations and an agreed method for their use are now available from listed centres as ICSH/ISBT and Food and Drug Administration reference materials.

Blood Grouping and Crossmatching↗
PubMed1994

Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal differentiation.

The NF-kappa B/Rel family of at least five transcription factor polypeptides is thought to function both as a developmental regulator in B cells and as a rapid response system in all cells. To examine this notion in more detail, we determined the protein contents of both the inducible and constitutive NF-kappa B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI 231. NF-kappa B p50/p65 is the major inducible nuclear complex after lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The constitutive and inducible complexes in WEHI 231 cells are mainly composed of p50 and Rel. The constitutive or induced activities are all sensitive to I kappa B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting that the balance between synthesis and degradation of I kappa B-alpha determines whether a particular cell lineage has constitutive activity. A patterned expression of the NF-kappa B/Rel activator proteins emerges from an analysis of other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may reflect the requirement for expression of different genes during terminal B-cell differentiation because different combinations of NF-kappa B/Rel family members preferentially activate distinct kappa B sites in reporter constructs.

Animals↗
PubMed1993

Production of high-titer helper-free retroviruses by transient transfection.

The generation of high-titer, helper-free retroviruses by transient transfection has been achieved by using the highly transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packaging cell line BOSC 23 produces infectious retrovirus at > 10(6) infectious units/ml of supernatant within 72 hr after CaPO4-mediated transfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as well as be useful for gene therapy.

3T3 Cells↗
PubMed1993

Quantitation of monoclonal antibodies by ELISA. The use of purified mouse IgG and mouse IgM monoclonal antibodies as standards in a quantitative ELISA measuring monoclonal antibodies produced by cell culture.

Murine monoclonal antibodies (MAbs) of IgG1, IgG2a and IgG2b subclasses and IgM class in hybridoma culture supernatants were quantified using a sensitive, reliable, optimized indirect double antibody sandwich ELISA. In the ELISA, the MAb in the culture supernatants was sandwiched between affinity isolated heavy chain specific polyclonal antibodies used for capture and detection. Quantitation was achieved by comparison with a standard curve produced by a purified MAb of the same class, subclass or ideally the same clone as the MAb to be quantified. These quantitative results were compared with those obtained using purified IgG and IgM polyclonal serum samples as standards and those obtained by total protein estimation using measurement at OD280nm. The IgG subclass MAbs used as standards were purified using protein G and the IgM class MAb was purified by ion exchange followed by gel filtration chromatography. Bovine IgG contamination of the MAb supernatants and the purified MAbs was also measured by a double antibody sandwich ELISA.

Animals↗
PubMed1993

The p65 subunit of NF-kappa B regulates I kappa B by two distinct mechanisms.

Transcription factor NF-kappa B (p50/p65) is generally localized to the cytoplasm by its inhibitor I kappa B. Overproduced I kappa B, free from NF-kappa B, is rapidly degraded. Overexpression of p65 increases endogenous I kappa B protein in both carcinoma and lymphoid cells by two mechanisms: protein stabilization and increased transcription of I kappa B mRNA. In contrast, p65 delta, a naturally occurring splice variant, fails to markedly augment I kappa B protein levels. Both overexpressed p65 and coexpressed p50 are cytoplasmic, whereas p65 delta is partly nuclear, indicating that the I kappa B induced by p65 can maintain NF-kappa B in the cytoplasm. Thus, p65 and I kappa B are linked in an autoregulatory loop, ensuring that NF-kappa B is held in the cytoplasm until cells are specifically induced to translocate it to the nucleus.

3T3 Cells↗
PubMed1993

The candidate proto-oncogene bcl-3 encodes a transcriptional coactivator that activates through NF-kappa B p50 homodimers.

The candidate proto-oncogene bcl-3 encodes a protein that shares structural features with I kappa B-alpha and other proteins that bind to members of the Rel protein family. Here, we show that in contrast to the inhibitory activity of I kappa B-alpha, the bcl-3 gene product superactivates NF-kappa B p50 homodimer-mediated gene expression both in vivo and in vitro. BCL-3 protein can, as well, selectively associate with p50 homodimers in the presence of DNA containing a kappa B motif. These results strongly suggest that BCL-3 can act as a transcriptional coactivator, acting through DNA-bound p50 homodimers.

Animals↗
PubMed1993

The bcl-3 proto-oncogene encodes a nuclear I kappa B-like molecule that preferentially interacts with NF-kappa B p50 and p52 in a phosphorylation-dependent manner.

The product of the putative proto-oncogene bcl-3 is an I kappa B-like molecule with novel binding properties specific for a subset of the rel family of transcriptional regulators. In vitro, Bcl-3 protein specifically inhibited the DNA binding of both the homodimeric NF-kappa B p50 subunit and a closely related homolog, p52 (previously p49), to immunoglobulin kappa NF-kappa B DNA motifs. Bcl-3 could catalyze the removal of these proteins from DNA. At concentrations that significantly inhibited DNA binding by homodimeric p50, Bcl-3 did not inhibit binding of reconstituted heterodimeric NF-kappa B (p50:p65), a DNA-binding homodimeric form of p65, or homodimers of c-Rel. Phosphatase treatment of Bcl-3 partially inactivated its inhibitory properties, implicating a role for phosphorylation in the regulation of Bcl-3 activity. Bcl-3, like p50, localizes to the cell nucleus. In cells cotransduced with Bcl-3 and p50, both molecules could be found in the nucleus of the same cells. Interestingly, coexpression of Bcl-3 with a p50 mutant deleted for its nuclear-localizing signal resulted in the relocalization of Bcl-3 to the cytoplasm, showing that the proteins interact in the cell. These properties contrast Bcl-3 to classically defined I kappa B, which maintains heterodimeric NF-kappa B p50:p65 in the cytoplasm through specific interactions with the p65 subunit. Bcl-3 appears to be a nuclear, I kappa B-related molecule that regulates the activity of homodimeric nuclear p50 and its homolog p52.

Animals↗
PubMed1993

In vitro insulin-like growth factor-I, growth hormone, and insulin resistance occurs in symptomatic human immunodeficiency virus-1-infected children.

Poor growth is a common feature of symptomatic children (Centers for Disease Control stage P2) infected with human immunodeficiency virus-1 (HIV-1). However, several previous studies have failed to show any relationship between serum hormone levels and poor growth. To assess the roles of hormone deficiency and hormone resistance in the development of poor growth in HIV-1-infected children, we studied six asymptomatic Centers for Disease Control stage P1 [height SD score = 0.01 +/- 1.0 (mean +/- SD)], 10 P2 (height SD score = -2.0 +/- 1.0), and six short, normal children (height SD score = -2.4 +/- 1.2). Mean weight:height SD scores were similar in all three groups, suggesting that gross nutritional status did not differ between groups. There were no significant differences between groups with respect to mean plasma levels of IGF-I, thyroid hormones, TSH, and cortisol. As an index of hormone sensitivity, we quantified in vitro colony formation of erythroid progenitor cells, isolated from peripheral blood of study subjects, in response to IGF-I, growth hormone (GH), and insulin. P2 subjects had a quantitative mean reduction in erythroid progenitor cells colony formation in response to IGF-I of 32% compared with P1 subjects (p = 0.001 by analysis of variance) and 21% compared with controls (p = 0.006); in response to GH of 21% compared with controls (p = 0.015); and in response to insulin of 35% compared with P1 subjects (p = 0.038) and 34% compared with controls (p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)

Child↗
PubMed1992

Prevalence of three mutations in the Gs alpha gene among 24 families with pseudohypoparathyroidism type Ia.

Pseudohypoparathyroidism type Ia (PHP-Ia), an inherited multi-hormone resistance syndrome, is associated with deficient cellular activity of the alpha-subunit of the guanine nucleotide-binding protein (Gs alpha) that stimulates adenylyl cyclase. We determined prevalence of three recently described mutations in exons 1 and 10 of the Gs alpha gene among 24 unrelated patients with PHP-Ia. Restriction analysis was used to detect two mutations that produce unique RFLPs, and allele-specific oligonucleotide hybridization was used to detect the other mutation. As none of these mutations were not found, genomic DNA was analyzed with denaturing gradient gel electrophoresis to screen for other mutations in exon 10. Mutations of the initiation codon and exon 10 in the Gs alpha gene thus rarely (< or = 4% each) cause PHP-Ia and the Gs alpha gene mutations causing PHP-Ia are heterogeneous and unique to each pedigree.

Adenylyl Cyclases↗
PubMed1992

Recovery of consummatory feeding behavior after bilateral lesions of the cerebral-buccal connectives in Aplysia californica.

In the sea hare, Aplysia californica, consummatory feeding behavior is selectively abolished by bilateral crushes of the cerebral-buccal connectives and recovers by postlesion day 13. Recovered biting responses are initially weak and increase in magnitude gradually with time. The lesions do not affect appetitive feeding behavior or unrelated reflexive behaviors. Thus, feeding in Aplysia can be used to examine the neural basis of behavioral recovery after CNS injury.

Animals↗
PubMed1992

Prediction of response to lithotripsy--the use of scanning electron microscopy and X-ray energy dispersive spectroscopy.

A single urine sample was obtained from each of 38 patients before and immediately after treatment with a Wolf Piezolith 2300. Each sample was analysed for crystals using scanning electron microscopy and X-ray energy dispersive spectroscopy. In all cases it was possible to make a stone diagnosis prior to treatment and there was 100% correlation between the pre- and post-treatment diagnosis. All stones smaller than 90 mm2 on plain X-ray cleared with piezoelectric lithotripsy alone, regardless of composition. Larger stones composed of calcium oxalate were all successfully fragmented, but 5 of 15 mixed or phosphate stones failed to respond.

Adolescent↗
PubMed1991

Neuromuscular organization of the buccal system in Aplysia californica.

The intrinsic muscles and peripheral nerves in the buccal system of the sea hare Aplysia californica were studied to build a foundation on which to base future investigations of feeding in intact animals. A detailed description of the bilaterally paired intrinsic muscles is given identifying previously unreported muscles. Each of the six buccal nerves (n1-n6) and the cerebrobuccal connective (CBC) have been characterized in several respects. Cell bodies in the buccal ganglion with projections into each of the buccal nerves have been identified via the cobalt backfilling technique. All nerves contain axons of cell bodies in the ipsilateral as well as the contralateral ganglia. For each nerve, there is a consistent pattern in the distribution of cell bodies in the paired ganglia with the number of cell bodies in the contralateral ganglion being less than or equal to the number in the ipsilateral ganglion. Although the total number of backfilled cell bodies varies among the nerves, their size ranges are similar with the majority being small. Nerves 1, 2, 4, 5, and 6 provide motor innervation to the intrinsic buccal muscles in varying degrees with nerve 4 supplying all the intrinsic muscles; nerve 2 supplies only one. The axon composition of each nerve was scrutinized and revealed large numbers of axon profiles, the majority of which were less than 2 microns in diameter. The present study provides a framework for analysis of feeding behavior in Aplysia californica.

Animals↗