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Biomedical subjects

R Sack

Publications and source records attributed to R Sack.

At least 19 recordsLinked to original sources
PubMed2001

Target gene search for the metal-responsive transcription factor MTF-1.

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding alpha-fetoprotein, the liver-enriched transcription factor C/EBPalpha and tear lipocalin/von Ebner's gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns gamma-glutamylcysteine synthetase(hc) (gamma-GCS(hc)), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. gamma-GCS(hc) mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

Animals↗
PubMed2001

Effect of external ocular surgery and mode of post-operative care on plasminogen, plasmin, angiostatins and alpha(2)-macroglobulin in tears.

PURPOSE: To determine whether corneal surgery and the mode of post-surgical treatment influence the distribution of plasminogen, plasmin, angiostatins and alpha(2)-macrogobulin in tear fluid. METHODS: Subjects underwent either photorefractive keratectomy (PRK), insertion of intra-stromal corneal rings (ICR), or cataract ablation followed by insertion of an intra-ocular lens (IOL). Post-surgical treatment consisted of prophylactic use of antibiotic and anti-inflammatory agents followed either by patching for 24 hours, or covering the wounded cornea with a bandage soft contact lens. Open eye tear fluid (OTF) was obtained prior to surgery and 10 minutes after patch removal or 24 hours after surgery and thereafter with the bandage lens still in place. After centrifugation, supernatants and controls were western blot analyzed using a protocol designed to allow the simultaneous semi- quantitative detection of alpha2-macroglobulin, plasminogen, plasmin, angiostatins and interleukin-8 (IL-8). RESULTS: No obvious differences were apparent in OTF recovered from contralateral control eyes compared to the surgical eyes in individuals who underwent PRK surgery and whose eyes were covered with a bandage contact lens. In contrast, OTF samples recovered 10 minutes after patch removal from all individuals contained elevated levels of alpha2-macroglobulin and a diverse mixture of elevated levels of plasminogen/plasmin, angiostatins and possibly a plasmin-a1-antiplasmin complex. All of these changes were seen, albeit to a lesser extent, in the patched control OTF samples. IL-8 could not be detected in any sample. The composition of the tear film returned to near normal on subsequent sampling 24 hours after patch removal. CONCLUSIONS: Patching results in a marked increase in the concentration of various proteins which could modulate inflammation and wound healing.

Angiostatins↗
PubMed2001

Identification, cloning and characterisation of a novel copper-metallothionein in tetrahymena pigmentosa. Sequencing of cDNA and expression.

The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the induction of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducible MT-isoform isolated from Tetrahymena after exposure to a non-toxic dose of copper. The cDNA sequence was determined utilising the partial peptide sequence of purified protein. The Cu-MT cDNA encodes 96 amino acids containing 28 cysteine residues (29%) arranged in motifs characteristic of the metal-binding regions of vertebrate and invertebrate MTs. Both the amino acid and nucleotide sequences differ, not only from other animal MTs, but also from the previously characterised Tetrahymena Cd-MT. Both MTs contain the structural pattern GTXXXCKCXXCKC, which may be proposed as a conservative sequence of Tetrahymena MTs. Cu-dependent regulation of MT expression was also investigated by measuring MT-mRNA and MT levels. MT synthesis occurs very quickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological functions, are expressed according to the inducing metal, Cu or Cd.

Amino Acid Sequence↗
PubMed2000

Isolation and characterization of a self-sufficient one-domain protein. (Cd)-metallothionein from Eisenia foetida.

Earthworms have been shown to accumulate trace elements in general, and particularly high amounts of metal ions such as cadmium, copper and zinc. The earthworm's response to metal contamination has been linked to the induction and expression of metallothionein (MT) proteins, a detoxification strategy analogous to that found in other biological systems. The present study focuses on an inducible Cd-MT isolated from the compost-dwelling brandling worm Eisenia foetida (Savigny). A full characterization of the protein (including protein induction, MT cDNA, amino-acid sequence and metal stoichiometry) revealed a new dimension of knowledge to the molecular genetic information available to date. Whereas the elucidated cDNA codes for a putative protein which possesses 80 amino-acid residues, the characterized protein bears only 41 amino acids. The isolated product has evidently attained its size and shape by cleavage near the N-terminal site and at the linker region between the two putative metal-binding domains of the translated product, yielding a small MT moiety which contains 12 Cys residues (including one triple Cys-motif) binding four cadmium ions. It can be shown that the isolated MT molecule represents a self-sufficient one-domain MT which is stable in vitro. The isolation of the single-domain MT peptide raises the question about the method of formation and significance in vivo of such small MT moieties from tissues of E. foetida and possibly other terrestrial invertebrates. In this respect, two hypotheses are discussed: firstly, the possibility of formation of small MT peptides due to enzymatic cleavage of the intact protein during the process of preparation and isolation; and secondly, the possibility of deliberate post-translational processing of the translated gene product to yield functional one-domain MT moieties.

Amino Acid Sequence↗
PubMed1999

Spontaneous deamidation and isomerization of Asn108 in prion peptide 106-126 and in full-length prion protein.

In prion-related encephalopathies, the cellular prion protein (PrP(C)) undergoes a change in conformation to become the scrapie prion protein (PrP(Sc)) which forms infectious deposits in the brain. Conceivably, the conformational transition of PrP(C) to PrP(Sc) might be linked with posttranslational alterations in the covalent structure of a fraction of the PrP molecules. We tested a synthetic peptide corresponding to residues 106-126 of human PrP for the occurrence of spontaneous chemical modifications. The only asparagine residue, Asn108, was deamidated to aspartic acid and isoaspartic acid with a half-life of about 12 days. The same posttranslational modifications were found in recombinant murine full-length protein. On aging, 0.8 mol of isoaspartyl residue per mole of protein was detected by the protein-l-isoaspartyl methyltransferase assay (t(1/2) approximately 30 days). Mass spectrometry and Edman degradation of Lys-C fragments identified Asn108 in the amino-terminal flexible part of the protein to be partially converted to aspartic acid and isoaspartic acid. A second modification was the partial isomerization of Asp226' which is only present in rodents.

Amino Acid Sequence↗
PubMed1992

Residual hydrogen peroxide as a function of platinum disc age.

Recently there has been considerable attention given to the possible deleterious effects of residual hydrogen peroxide on both the cornea and the crystalline lens of the eye. We measured residual hydrogen peroxide levels of the AOSept disinfection system at regular intervals over a 4 month period. By 1 month the residual hydrogen peroxide level was 21 ppm +/- 9.4 (mean +/- standard deviation); at the end of 2 months it was 36 ppm +/- 17.6. At 3 months, when disc replacement is recommended, the residual hydrogen peroxide level was 43 ppm +/- 4.7 (range: 17-98 ppm). At 4 months the residual hydrogen peroxide level rose to a mean of 48 ppm +/- 18.2. The increase in measured residual hydrogen peroxide between month 1 and each successive month was found to be statistically significant. However, these levels failed to induce an increase in symptomatology or slit lamp findings. As determined by scanning electron microscopy there was an erosion of the platinum coating with time that appeared to coincide with the measured residual peroxide level. Cultures of randomly selected platinum discs at the end of 4 months failed to reveal more than isolated positive findings of Staphylococcus epidermidis. While an increase in residual peroxide with time may lead to greater disinfecting capabilities of this system, the possibility of corneal damage as a direct result of high peroxide levels is of concern.

Adult↗
PubMed1989

[Gas chromatography studies of exhaust air from an animal body waste salvage industry].

The proceedings of animal body waste salvage plants are-as you know-connected with intense smell importunities of the immediate environment. Suited desodorization plants are required to limit this problem of the environmental hygiene. The presupposition for a high effectiveness of these plants is to know the qualitative composition of the exhaust air, the analysis of which is the subject of the article in hand.

Abattoirs↗
PubMed1987

Comparative diets of a idiopathic senile cataract and normal population: dietary risk factors in cataractogenesis.

A survey was carried out contrasting the diets of population with senile cataracts and control populations. Results failed to confirm the reports of others suggesting that ingestion of moderate amounts of seafood containing mercury or excess amounts of milk and milk products represent risk factors for senile cataractogenesis. Several dietary differences were noted lending credence to the prophylatic values of riboflavin enrichment to decrease risk of cataractogenesis and suggesting areas for further study.

Aged↗
PubMed1984

Grand mal discharge.

Two hundred nineteen grand mal discharges were reviewed in 50 epileptic patients. It was commonly seen in patients with prenatal and perinatal causes, mental deficiency with neurological deficits, and multiple types of seizures. Eighty-eight percent of the discharges were bilaterally synchronous with 49 percent of bifrontal predominance. Fifty-eight percent were associated with drowsiness or sleep, and 64 percent were associated with clinical accompaniments. The most common clinical signs were tonic movement of arms, legs or neck, eye opening, eye fluttering, head drop, etc. The increased frequency of clinical seizures usually observed during the occurrence of this pattern may provide some clues that the antiepileptic medication should be reassessed to control the oncoming seizure attacks.

Adolescent↗
PubMed1982

Single unit activity in the rat's midbrain during nocifensive tail flick reaction.

Single units have been recorded extracellularly from the periaqueductal central grey and from adjacent structures in behaving rats during tail flick reflexes elicited by controlled radiant heat. Together with the spike recordings, the heart rate of the animal and a thoracic myogram as an indicator of overall motor activity, have been recorded. Twenty-seven medial midbrain units have been studied, which were all characterized by being spontaneously active and by receiving input from myelinated afferent fibres from the tail, as shown by weak transcutaneous electrical stimulation. These units showed a variety of changes in discharge pattern. The most frequently observed change in activity was an increase of firing in the pre-flick period (10 of 27 units) and a decrease of firing below the prestimulus level in the post-flick period (12 of 27 units). It is suggested that changes in the discharge patterns were more closely related to behavioral output than to the input side of nociceptive behaviour.

Animals↗
PubMed1977

The indoleamine hypothesis of depression: an overview and pilot study.

This paper reviews the evidence for a specific indoleamine deficiency in depression and the attempts to correct this suspected deficiency with serotonin precursors. It also presents the clinical and biochemical data on six patients with depression treated with L-5-HTP in a nonrandom, double-blind protocol. The oral administration of L5-HTP was associated with a rise in CSF5-HIAA, but only two of six patients studied had any decrease in depression ratings. 5-HTP was also shown to decrease urinary excretion of 17 hydroxycorticosteroids in twodepressed patients and three normal controls suggesting an interrelationship between serotonin metabolism and the pituitary adrenal system. This leads to the suggestion that in a postulated subgroup of depressed patients with pituitary adrenal hyperactivity and evidence of serotonin deficiency, L5-HTP deserves a further trial as an experimental treatment.

5-Hydroxytryptophan↗