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Biomedical subjects

G Baum

Publications and source records attributed to G Baum.

At least 19 recordsLinked to original sources
PubMed1996

Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

Calmodulin↗
PubMed1996

Activation of a recombinant petunia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody which recognizes the calmodulin binding domain.

To date, only plants have been shown to possess a form of glutamate decarboxylase (GAD) that binds calmodulin. In the present study, a recombinant calmodulin-binding 58-kDa petunia GAD produced in Escherichia coli was purified to homogeneity using calmodulin-affinity chromatography, and its responsiveness to calcium and calmodulin was examined in vitro. At pH 7.0-7.5, the purified recombinant enzyme was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity (Vmax = 30 micromol of CO2 min-1 mg of protein-1) by the addition of exogenous calmodulin (K0.5 = 15 nM) in the presence of calcium (K0.5 = 0.8 microM). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (Km = 8.2 mM). A monoclonal antibody directed against the carboxyl-terminal region, which contains the calmodulin-binding domain of GAD, was able to fully activate GAD in a dose-dependent manner in the absence of calcium and calmodulin, whereas an antibody recognizing an epitope outside of this region was unable to activate GAD. This study provides the first evidence that the activity of the purified 58-kDa GAD polypeptide is essentially calcium/calmodulin-dependent at physiological pH. Furthermore, activation of GAD by two different proteins that interact with the calmodulin-binding domain, a monoclonal antibody or calcium/calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.

Amino Acid Sequence↗
PubMed1996

Small bowel fistulas.

The treatment of small bowel fistulas remains a difficult problem. Spontaneous closure rates of 30% are currently being achieved after 4 to 6 weeks of conservative therapy with bowel rest, TPN, octreotide, and control of sepsis. Resection of the fistulous bowel is warranted if fistula closure has not occurred by 4 to 6 weeks.

Enteral Nutrition↗
PubMed1995

Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase.

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.

Amino Acid Sequence↗
PubMed1993

A plant glutamate decarboxylase containing a calmodulin binding domain. Cloning, sequence, and functional analysis.

Molecular procedures have been applied to isolate plant calmodulin-binding proteins. A petunia cDNA expression library was screened with 35S-labeled recombinant calmodulin as a probe, and a cDNA coding for a Ca(2+)-dependent calmodulin-binding protein was isolated. The deduced amino acid sequence of the petunia protein (500 amino acid residues, 58 kDa) has 67% overall amino acid sequence similarity to glutamate decarboxylase (GAD) from Escherichia coli (466 amino acid residues, 53 kDa). The recombinant protein expressed in E. coli cells displays GAD activity, i.e. catalyzes the conversion of glutamic acid to gamma-aminobutyric acid and binds calmodulin, whereas E. coli GAD does not bind calmodulin. The calmodulin binding domain in the petunia GAD was mapped by binding truncated forms of GAD immobilized on nitrocellulose membranes to recombinant petunia 35S-calmodulin as well as to biotinylated bovine calmodulin and by binding truncated forms of GAD to calmodulin-Sepharose columns. The calmodulin binding domain in petunia GAD is part of a carboxyl end extension that is not present in E. coli GAD. Polyclonal antibodies raised against the recombinant petunia GAD detect a single protein band from plant extracts of gel mobility identical to that of the recombinant GAD. Moreover, the plant protein binds calmodulin in vitro. This is the first report of the isolation of a GAD gene from plants and of a calmodulin-binding GAD from any organism. Our results raise the possibility that intracellular Ca2+ signals via calmodulin are involved in the regulation of gamma-aminobutyric acid synthesis in plants.

Amino Acid Sequence↗
PubMed1991

Clarification of ascites fluid.

An essential first step in the purification of monoclonal antibodies is the clarification of the ascites fluid. While not ignored, this step has not received critical attention. Six different procedures were evaluated with respect to effectiveness and impact on several selected chemical components. For large-scale work, we find an on-line filtration process to be most efficient.

Animals↗
PubMed1990

Regulation of tropomyosin expression in transformed granulosa cell lines with steroidogenic ability.

The expression of the different tropomyosin isoforms was analyzed in primary granulosa cell cultures and in established granulosa cell lines cotransfected with SV40 and Ha-ras DNA which retain a high steroidogenic response to cAMP stimulation. In contrast to normal cells which greatly reduce the expression of all tropomyosin isoforms during development of steroidogenic ability, in the doubly transformed cells only the synthesis of the high molecular weight isoforms nos 2 and 3 was decreased. The expression of isoforms 1 and 5 was elevated in the cotransfected lines and that of tropomyosin 1 was further enhanced by cAMP stimulation. The increased synthesis of tropomyosins 1 and 5 is unique to SV40 transformation, since it was observed also in cells transfected with SV40 DNA alone. These cells displayed a well organized microfilament system, but have lost the ability to differentiate. The reduced expression of tropomyosins 2 and 3 and a poorly organized microfilament system appear to be a dominant feature of both the highly differentiated normal- and transformed-granulosa cells. It is suggested that the switches in tropomyosin isoform expression during development of the steroidogenic phenotype and in cell transformation may account for necessary changes in microfilament organization which accompany these cellular processes.

Actins↗
PubMed1989

Regulation of tropomyosin expression in the maturing ovary and in primary granulosa cell cultures.

Granulosa cell differentiation in vitro in response to gonadotropins is characterized by major changes in cell shape, cell aggregation, and the organization of microfilaments. These changes are associated with enhanced steroidogenesis in maturing granulosa-lutein cells. Since nonmuscle tropomyosin isoforms were implicated in stabilizing actin filaments, we studied the organization and expression of tropomyosin in differentiating primary cultures of rat granulosa cells and during ovarian folliculogenesis and luteinization. In unstimulated primary granulosa cell cultures tropomyosin was found mainly along stress fibers. In differentiating cells tropomyosin staining was diffuse with sometimes a subcortical organization. The changes in tropomyosin organization were accompanied by a pronounced decrease in the synthesis, translation in vitro, and mRNA levels of all the rat nonmuscle tropomyosin isoforms, with a greater reduction in the higher molecular weight isoforms than in the smaller isoforms. Similar results were obtained whether cells were stimulated to differentiate with gonadotropins, with cAMP, by culturing cells on an extracellular matrix, or by treatment with cytochalasin B. The effect of cytochalasin B was reversible; upon removal of the drug tropomyosin synthesis increased to near control levels, while that of proteins associated with luteinization decreased drastically. RNA isolated from ovaries with follicles at the preantral, preovulatory stage and from corpora lutea contained decreased tropomyosin mRNA levels during ovarian luteinization when the level of RNA for a key steroidogenic enzyme, cytochrome P-450 cholesterol side chain cleavage (P-450 scc), increased. The results suggest a physiological relevance for the low level of tropomyosin expression in the mechanisms which bring about the morphological and biochemical development and maturation of granulosa cells.

Actin Cytoskeleton↗
PubMed1988

Rehabilitation of the patient with chronic back pain. A search for outcome predictors.

This prospective study was initiated 3 years ago to evaluate the outcome and to identify predictors of success or failure in patients admitted to a rehabilitation program for chronic low-back pain. Multiple parameters were evaluated, including psychologic data (MMPI, personal interview, pain drawing, etc.), physical measurements (flexibility, strength and endurance), and demographic data concerning the patient's home and working environment. Information was available on each patient admitted to the program prior to his admission, at completion of the program, 6 weeks following completion of the program and 3 months following completion of the program. A telephone interview was carried out 2 1/2 years following the patient's discharge from the program. Linear regression analysis was used to identify the important independent variables with regard to the dependent variables of relief of back pain, return to work and increased activities at home. Demographic data were of no value as a predictor with the exception of age and returning to work. The patients over the age of 50 returned to work with much less frequency than those less than 50. Psychologic information from the MMPI and similar tests were of no value. The personal preadmission interview of a trained psychologist, however, was a good predictor of an individual's eventual return to work and overall improvement. Worker's Compensation and other litigation was a negative factor in a patient's prognosis. The treatment team's prognosis at the time of discharge from the program was the best overall predictor of a patient's chance of success or failure in the longterm.

Activities of Daily Living↗
PubMed1986

The role of protein metabolism in 204 liver cirrhotics with and without hepatic encephalopathy. II. Amino acids, free phenols and indoles.

Toxic protein metabolites are assumed to play an important role in the multifactorial pathogenesis of hepatic encephalopathy (HE). To investigate this, we examined the serum levels of free amino acids, free phenols and indoles in 100 healthy adults, and in 124 liver cirrhotics with HE and 80 without HE. We found a significant increase in free serum phenols and indican already in liver cirrhosis without portal hypertension (PH) and HE. In stage III and IV HE large amounts of p-hydroxy-phenyl lactic acid were detected, which was not the case in cirrhotics without HE. In HE the increase in free serum phenols and indican was much higher than that of the mother substances tyrosine and tryptophan. The quotient BCAA/AAA was decreased significantly already in PH without HE. In addition to the increased formation by intestinal bacteria, a diminished oxidative capacity of the cirrhotic liver seems to be one of the main causes of the increased serum levels of toxic protein metabolites in HE.

Adult↗