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Biomedical subjects

H Fromm

Publications and source records attributed to H Fromm.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1993

Formation, absorption, and biotransformation of delta 6-lithocholenic acid in humans.

delta 6-Lithocholenic acid was identified in small amounts in fecal samples in vitro after incubation with ursodeoxycholic acid and in vivo in controls and after chenodeoxycholic and ursodeoxycholic acid ingestion. Fourteen to 45.0% of delta 6-[24-14C]lithocholenic acid was biotransformed in vitro in feces within 30 s. After colonic instillation of delta 6-[24-14C]lithocholenic acid, 50% of the radioactivity appeared in bile acids, most of it in lithocholic acid, within 3 h. Jejunal perfusions with delta 6-[24-14C]lithocholenic acid showed 33-92% absorption. One hour after jejunal instillation of 1 mmol, 4.4-27.5% of the biliary radioactivity was found in ursodeoxycholic, chenodeoxycholic, lithocholic, and 7-ketolithocholic acids. A sulfated glycine conjugate of delta 6-lithocholenic acid was identified in bile. One hour after intravenous injection of delta 6-[24-14C]lithocholenic acid, 40.1-42.6% of biliary radioactivity appeared in 7-ketolithocholic, chenodeoxycholic, lithocholic/isolithocholic, and ursodeoxycholic acids. The studies show that delta 6-lithocholenic acid is 1) formed in colonic lumen from chenodeoxycholic and ursodeoxycholic acids, 2) well absorbed in small intestine, and 3) biotransformed in both the colonic lumen and liver. The studies also identified delta 6-lithocholenic acid as a new intermediate in formation of lithocholic acid. Finally, the studies showed that a small portion of delta 6-lithocholenic acid is excreted as a sulfated glycine conjugate in bile.

Bile↗
PubMed1993

Ursodeoxycholate mobilizes intracellular Ca2+ and activates phosphorylase a in isolated hepatocytes.

In isolated hamster hepatocytes, ursodeoxycholic acid (UDCA) mobilized intracellular free calcium ([Ca2+]i) and activated phosphorylase a with a half-maximally effective concentration of 188 and 9 microM, respectively. Addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) did not affect the maximum [Ca2+]i mobilized by UDCA; however, [Ca2+]i returned to basal levels in 4-5 min compared with > 10 min in the absence of EGTA. Both UDCA and vasopressin activated phosphorylase a to the same extent in the presence and absence of extracellular Ca2+, and the effect of both agents was abolished when the cells were depleted in Ca2+. Vasopressin (100 nM) did not further mobilize [Ca2+]i or activate phosphorylase a when combined with 500 microM UDCA. However, unlike vasopressin, UDCA did not stimulate inositol 1,4,5-trisphosphate (IP3) formation. In contrast to taurine-conjugated UDCA (TUDCA), concentration < or = 500 microM of glycine-conjugated UDCA (GUDCA) did not affect either [Ca2+]i or phosphorylase a. Lithocholic acid and taurolithocholic acid (TLCA) displayed the highest affinity for Ca2+. In addition, TLCA, chenodeoxycholic acid, and NaF stimulated Ca2+ efflux at concentrations as low as 100 microM, 200 microM, and 5 mM, respectively. Conversely, UDCA, TUDCA, and GUDCA presented the lowest affinity for Ca2+ and had no effect on Ca2+ efflux. The 28% increase in Ca2+ release induced by TLCA alone was further augmented to approximately 60% when TLCA was combined with UDCA, TUDCA, or GUDCA. However, Ca2+ efflux induced by NaF was not further increased by UDCA and its conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoquinolines↗
PubMed1993

Comparative binding of bile acids to serum lipoproteins and albumin.

Characteristics of the binding of lithocholic acid (LC), chenodeoxycholic acid (CDC), and cholic acid to human plasma proteins were studied. Affinity of the different plasma protein fractions for the bile acids studied decreased with increased polarity of the steroid nucleus of the bile acid. Binding of LC, CDC, and cholic acid to the lipoprotein-free, albumin-rich plasma fraction was characterized by two classes of binding sites with respective KDs of 2, 5, and 51 microM, and of 39, 2,387, and 5,575 microM, while corresponding Bmax values were similar for the different bile acids, at around 6 and 100 nmol/mg protein. Bile acid binding to the different lipoprotein fractions was characterized by a single population of binding sites, with a KD ranging from 47 to 66 microM for LC, 695 to 1010 microM for CDC, and 2,511 to 2,562 microM for cholic acid. Bmax values, at 416-913 nmol/mg protein, were similar among the different bile acids studied. Both glycine- and taurine-conjugated, as well as unconjugated LC competitively inhibited [24-14C]LC binding to low density (LDL) and to high density lipoproteins (HDL) to the same extent, while the more polar LC-3-sulfate, CDC, and cholic acid were increasingly less potent in displacing LC binding from the respective lipoproteins. Furthermore, all bile acids studied shared the same lipoprotein binding site. The lipoprotein fluorescence at 330-334 nm, following excitation at 280 nm, was diminished after incubation with LC, suggesting that the bile acid masks the tryptophan residues of the protein moiety. Finally, the initial rate of uptake of 1 microM LC, in isolated hamster hepatocytes, at around 0.045 nmol.sec-1.mg cell wt-1, was not affected by the protein carrier. However, for the same concentration of LC, bound to either LDL or HDL, LC binding resulted in 75-77% of the total [24-14C]LC nonspecifically bound to the hepatocyte, compared to 65% when bound to albumin, and 45% in the absence of protein. The studies show that, under conditions when the serum bile acid concentration exceeds the capacity of the high affinity class of albumin binding sites for bile acids, lipoproteins have similar or greater affinity to bind bile acids than does albumin. The ability of lipoproteins to increase the nonspecific association of lithocholic acid with liver cells may also facilitate bile acid association with extrahepatic tissues. As lipoproteins, in contrast to albumin, are targeted to most cells, they may play a major role in the transport of potentially toxic bile acids to peripheral cells.

Animals↗
PubMed1992

Fecal bile acid excretion and composition in response to changes in dietary wheat bran, fat and calcium in the rat.

The effect and possible interactive influence of different dietary amounts of wheat bran, fat and calcium on the fecal excretion, concentration and composition of bile acids was studied in Fischer-344 rats. The fecal bile acids were analyzed using gas-liquid chromatography. Dietary wheat bran increased both total bile acid excretion and fecal weight without changes in fecal bile acid concentration. The proportion of fecal hyodeoxycholic acid decreased with increasing dietary fiber, whereas that of lithocholic and deoxycholic acids increased significantly with fiber intake. The percent content of fecal chenodeoxycholic acid did not change. Increasing dietary fat led to an increase in bile acid excretion without changes in either fecal weight or bile acid concentration. In contrast, the level of dietary calcium did not affect the total excretion of bile acids. However, since calcium increased the fecal weight, it consequently diluted bile acids and decreased their fecal concentration. Dietary fat and calcium had no influence on fecal bile acid composition. There were no interactive effects of wheat bran, fat and calcium on fecal bile acids. The finding in this study that dietary fiber, fat and calcium induce significant changes in fecal bile acids may be of relevance to the potential of bile acids to promote carcinogenesis.

Animals↗
PubMed1992

Bile acid dissolution therapy of gallbladder stones.

Oral cholelitholytic bile acid therapy has become established treatment for selected patients with cholesterol gallstones. The treatment finds its clinical application both alone and in combination with ESWL. UDCA alone or, less commonly, a combination of this bile acid with CDCA is used. Optimal results can be expected only in carefully selected patients. Bile acid dissolution therapy is most successful in patients with radiolucent gallstones which are < or = 0.5 cm in diameter or are shown by OCG to be floating. Dissolution is seldom seen when the stones are > 1 cm in size. Cholelitholytic treatment in combination with ESWL yields optimal results in single radiolucent gallstones which are not greater than 2 cm. ESWL thus makes it possible to use medical treatment effectively in single 1-2 cm gallstones when bile acids alone would not be successful. Bile acid treatment is extremely safe, especially if UDCA is given without the addition of CDCA.

Chenodeoxycholic Acid↗
PubMed1992

Prevention and early detection of colorectal cancer.

Adherence to a low-fat, high-fiber diet and regular screening to detect and identify polyps at an early, more treatable stage may reduce deaths from colorectal cancer. Patients should be told that they can reduce their risk of colorectal cancer by increasing their intake of fruits, vegetables and whole grain cereals and decreasing their intake of fat. Although scientific controversy exists regarding the specific methods and intervals of screening for colorectal cancer, family physicians should adopt a regular approach to screening in their patients. In adults 50 years of age or older, annual fecal occult blood testing is recommended. If positive results are obtained, follow-up examination should be performed with either colonoscopy or flexible sigmoidoscopy plus air-contrast barium enema. Persons between 50 and 75 years of age may also benefit from screening with flexible fiberoptic sigmoidoscopic examination every three to five years.

Aged↗
PubMed1991

Ursodeoxycholic acid increases low-density lipoprotein binding, uptake and degradation in isolated hamster hepatocytes.

Ursodeoxycholic acid (UDCA), in contrast to both chenodeoxycholic acid (CDCA), its 7 alpha-epimer, and lithocholic acid, enhanced receptor-dependent low-density lipoprotein (LDL) uptake and degradation in isolated hamster hepatocytes. The increase in cell-associated LDL was time- and concentration-dependent, with a maximum effect observed at approx. 60 min with 1 mM-UDCA. This increase was not associated with a detergent effect of UDCA, as no significant modifications were observed either in the cellular release of lactate dehydrogenase or in Trypan Blue exclusion. The effect of UDCA was not due to a modification of the LDL particle, but rather was receptor-related. UDCA (1 mM) maximally increased the number of 125I-LDL-binding sites (Bmax.) by 35%, from 176 to 240 ng/mg of protein, without a significant modification of the binding affinity. Furthermore, following proteolytic degradation of the LDL receptor with Pronase, specific LDL binding decreased to the level of non-specific binding, and the effect of UDCA was abolished. Conversely, the trihydroxy 7 beta-hydroxy bile acid ursocholic acid and its 7 alpha-epimer, cholic acid, induced a significant decrease in LDL binding by approx. 15%. The C23 analogue of UDCA (nor-UDCA) and CDCA did not affect LDL binding. On the other hand, UDCA conjugated with either glycine (GUDCA) or taurine (TUDCA), increased LDL binding to the same extent as did the free bile acid. The half maximum time (t1/2) to reach the full effect was 1-2 min for UDCA and TUDCA, while GUDCA had a much slower t1/2 of 8.3 min. Ketoconazole (50 microM), an antifungal agent, increased LDL binding, but this effect was not additive when tested in the presence of 0.7 mM-UDCA. The results of the studies indicate that, in isolated hamster hepatocytes, the UDCA-induced increase in receptor-dependent LDL binding and uptake represents a direct effect of this bile acid. The action of the bile acid is closely related to its specific structural conformation, since UDCA and its conjugates are the only bile acids shown to express this ability thus far. However, certain agents other than bile acids, such as ketoconazole, have a similar effect. Finally, the studies suggest that the recruitment of LDL receptors from a latent pool in the hepatocellular membrane may be the mechanism by which UDCA exerts its direct effect.

Animals↗
PubMed1991

[Ferritin in acute leukemia. Serum ferritin concentration as a nonspecific tumor marker for M1 and M2 myeloid leukemia].

Serum ferritin concentration was studied in 136 patients with different types of acute leukemia. Pretreatment serum ferritin concentrations in the immature myeloblastic leukemia (M1 and M2 of the FAB-classification of acute leukemias) was found to be highly increased compared to the more mature types of acute myeloblastic leukemias (M3 to M5) and the acute lymphoblastic leukemias (L1 to L3). Investigation of the intracellular ferritin concentration showed, that the serum ferritin levels paralleled the intracellular ferritin concentration within the leukemic blasts. Within the immature myeloic blasts (M1) the intracellular ferritin concentration was 14-fold increased compared to normal granulocytes. This correlated with the 17-fold increased serum ferritin levels in these patients. Intracellular ferritin concentrations within the leukemic blasts of more mature types of acute leukemia (M3 to M5) were found to be only slightly increased. These data support the concept, that an increased synthesis and release of ferritin by the leukemic blasts is responsible for the increased serum ferritin concentration. This concept is also supported by the observation, that a further increase of serum ferritin concentration was seen during a cytotoxic chemotherapy. It is noteworthy, that this increase was more pronounced in the immature leukemias obviously caused by a loss of intracellular ferritin from the damaged leukemic blasts. The serum ferritin levels followed closely the activity of the disease. Increased pretreatment serum ferritin concentrations normalized completely when patients achieved complete remission. In contrast, in patients with tumor relapse or tumor progression serum ferritin concentrations increased again. These data suggest that the serum ferritin in immature myeloblastic leukemia has the characteristics of a tumor associated marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers, Tumor↗
PubMed1991

Cerebral low-density lipoprotein (LDL) uptake is stimulated by acute bile drainage.

Although the cholesterol pool in the central nervous system is considered to be relatively stable, few studies have tested this assumption. The aim of the study was to gain further information on the communication between the extracerebral organs and the brain as far as cholesterol and lipoprotein transport are concerned. Receptor-dependent as well as receptor-independent LDL uptake in the brain were measured, by established methods, after constant 1-h intravenous infusions of [14C]sucrose-labelled hamster LDL and methylated human LDL, both in hamsters with an acute bile fistula and in control animals with an intact enterohepatic circulation. The receptor-dependent LDL uptake in the brain promptly showed a significant increase after the construction of the bile fistula. However, there was no difference in the receptor-independent LDL uptake between the bile fistula and control animals. The studies indicate the presence of close communications between extracerebral and brain cholesterol. Changes in the extracerebral compartments of cholesterol are, apparently, readily sensed by the LDL receptor in the brain and promptly evoke appropriate modifications in its activity.

Animals↗
PubMed1991

The tobacco transcription activator TGA1a binds to a sequence in the 5' upstream region of a gene encoding a TGA1a-related protein.

We have isolated and characterized a tobacco gene, designated G13, encoding a leucine zipper DNA-binding protein related to the transcription activator TGA1a. The G13 coding region is divided into eight exons and the amino acid sequence of the encoded protein (PG13) shows 76% homology to TGA1a. Their putative DNA-contacting regions (basic domains) are identical and they both bind to the same target sequences in vitro. By contrast, some differences are apparent between these proteins at the carboxyl end of the dimerization region (leucine zipper). The basic and leucine zipper domains are encoded on separate small exons. Analysis by DNAse I footprinting, gel shift and competition experiments revealed that TGA1a and PG13 synthesized in Escherichia coli, and the tobacco nuclear factor ASF-1 all bind to at least one site in the 5' upstream region of G13. The presence of a TGA1a binding site in the upstream region of a TGA1a-related gene suggests that transcription of this gene is autoregulated.

Amino Acid Sequence↗