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Biomedical subjects

Z Krawczyk

Publications and source records attributed to Z Krawczyk.

At least 19 recordsLinked to original sources
PubMed2006

Influx of macrophages into livers of rats treated with hepatotoxicants (thioacetamide, allyl alcohol, D-galactosamine) induces expression of HSP25.

Treatment of rats with a single dose of thioacetamide (TAA) provokes centrilobular inflammation and a significant expression of heat shock protein HSP25 in hepatocytes surrounding the area of inflammation. The HSP25 accumulation in hepatocytes adjacent to inflammatory regions was confirmed by identification of positive hepatocytes concentrated at periportal areas after treatment of rats with allyl alcohol (AA) or distributed diffusely throughout liver lobule after treatment with D-galactosamine (D-gal). In our model of TAA-treated rats the use of the anti-inflammatory drug-indomethacin, and the redox-regulating drug-N-acetylcysteine (NAC), significantly attenuated TAA-induced HSP25 expression and evoked morphological changes of recruited ED1+ macrophages. Treatment of rats with gadolinium chloride (GdCl(3)) decreased considerably the number of Kupffer cells (ED2+ macrophages) without affecting significantly the number and morphology of ED1+ macrophages as well as the expression pattern of TAA-induced HSP25. Our data shows for the first time that ED1+ macrophages recruited into the liver by treatment with TAA play a significant role in HSP25 induction in hepatocytes.

Animals↗
PubMed2006

Spermatocyte-specific expression of constitutively active heat shock factor 1 induces HSP70i-resistant apoptosis in male germ cells.

Spermatocytes, the most sensitive male germ cells to heat-induced apoptosis, do not respond to hyperthermia by inducing heat shock proteins (HSPs), including HSP70i, which has been previously shown to confer resistance to apoptosis in somatic cells. To dissect the mechanism of heat-induced apoptosis and to determine if we could protect spermatocytes by expressing HSP70i, we engineered transgenic mice that express in spermatocytes constitutively active heat shock transcription factor (HSF)1. Such HSF1 expression did not lead to transcription of inducible Hsp70 genes, but instead induced caspase-dependent apoptosis that mimicked heat shock-induced death of spermatogenic cells. Both mitochondria-dependent and death receptor-dependent pathways appear to be involved in such HSF1-induced apoptosis: the levels of Bcl-2 family proteins became increased, p53 protein accumulated and expression levels of caspase-8 and death-receptor-interacting proteins (including Fas-associated death domain protein and TNF receptor associated death domain protein) became elevated. Surprisingly, the constitutive spermatocyte-specific expression of HSP70i in double-transgenic males did not protect against such HSF1-induced apoptosis.

Animals↗
PubMed2003

Bovine lactoferrin decreases histopathological changes in the liver and regulates cytokine production by splenocytes of obstructive jaundiced rats.

OBJECTIVE AND DESIGN: The aim of this study was to evaluate effects of bovine lactoferrin (BLF) on histopathological changes in the liver of 14 day obstructive jaundiced (OJ) rats and production of tumor necrosis factor (TNF-alpha) and interleukin 6 (IL-6) by splenocytes from 7- and 14-day OJ rats. MATERIAL AND SUBJECTS: In the study 50 male rats of the Buffalo strain (170-270 g, mean 230 g) were used. TREATMENT: Rats were given 10 mg BLF in 0.5 ml saline daily, using a stomach tube. BLF was applied 2 days before operation and for 13 days following operation. METHODS: The specimens of liver were prepared using standard techniques. Sections, 5 mm thick were stained with hematoxylin and eosin and reviewed histologically. Microscopic estimation using semiquantitive 4-grade scale was used for evaluation of liver changes. For cytokine measurement splenocyte cultures were stimulated with 5 microg/ml lipopolysaccharide (LPS). After overnight incubation the activities of TNFalpha and IL-6 were determined using bioassays. For statistical evaluation of data the nonparametric Mann-Whitney test was applied. RESULTS: In rats with 14-day OJ, treated with BLF, the pathological changes in the liver were markedly reduced, in particular foci of necrosis with disseminated lymphocytes, cellular necrobiosis, bile duct proliferation and dilation. Neither proliferation of fibrous and reticular connective tissue nor activation of Kupffer cells was revealed. In the 7-day OJ rats treatment with BLF caused significant inhibition of both spontaneous (mean 253, median 275, vs mean 160, median 148 pg/ml, p=0.002) and LPS-induced TNF-alpha production (mean 4967, median 4102, vs mean 2291, median 2234 pg/ ml, p = 0.004) in the splenocyte cultures. The spontaneous (mean 120, median 81, vs mean 43, median 26 pg/ml, p=0.005) as well as LPS-induced IL-6 production (mean 422, median 378, vs mean 293, median 230 pg/ml, p = 0.025) were also lowered. On the other hand, in 14-day OJ, BLF upregulated cytokine production, in particular spontaneous (mean 148, median 158, vs mean 338, median 196, p = 0.001) and LPS-induced TNF-alpha (m ean 1331, median 1507, vs mean 2239, median 1707 pg/ml, p = 0.027). CONCLUSION: We conclude that oral administration of BLF is beneficial in alleviating deleterious effects of OJ.

Animals↗
PubMed2001

Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites.

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences.

5' Untranslated Regions↗
PubMed2001

Estimation of risk of inherited medullary thyroid carcinoma in apparent sporadic patients.

PURPOSE: The study was undertaken to evaluate the frequency of inherited medullary thyroid carcinoma (MTC) among patients with apparent sporadic disease. A stepwise algorithm was used depending on clinical indices and the age of patient at MTC diagnosis. PATIENTS AND METHODS: One hundred sixteen patients with MTC verified by postoperative pathologic examination were subjected to genetic analysis of RET exons 10, 11, 13, 14, and 16 by means of polymerase chain reaction, restriction endonuclease digestion, and DNA sequencing. RESULTS: Among 116 apparent sporadic MTC patients, we identified eleven (9.5%) RET germline mutation carriers. Seven of these (6.0%) were found by routine analysis (exons 10 and 11). The frequency of inherited disease among patients younger than 45 years at diagnosis was 10.2% by analysis of typical mutations in exons 10 and 11. Extended genetic analysis (sequencing of exons 11, 13, 14, and 16) yielded 6.1% additional diagnoses, giving a risk of 16.3% in this age group. One previously unreported mutation in exon 11 affected codon 649 (TCG>TTG, Ser>Leu). In the true sporadic MTC patients younger than 30 years at diagnosis, frequencies of 36% and 4.5% in polymorphic variants L769L and S836S, respectively, were observed. The frequency for L769L was higher than in older patients (P <.05). CONCLUSION: The frequency of inherited disease among apparent sporadic medullary thyroid carcinoma patients is close to 10% in the Polish population of MTC patients. The extended analysis of all known RET proto-oncogene mutation sites is obligatory in patients younger than 45 years at diagnosis, but we also see the need to analyze the impact of rarer mutations in older patients.

Adult↗
PubMed2000

Green fluorescent protein as a marker for monitoring activity of stress-inducible hsp70 rat gene promoter.

Murine melanoma cells B16(F10) were stably transfected with a plasmid containing GFP gene linked to rat stress-inducible hsp70.1 gene promoter. Transfected cells show in vitro variable basal levels of fluorescence depending on stress response induced at physiological temperature by growth conditions. Lack of manipulations except medium change resulted in reduction of cellular fluorescence. GFP expression in experimental murine tumors dropped to levels undetectable at physiological temperature. Heat shock induced significant fluorescence of tumor cells both in vitro and in vivo. GFP protein could be a useful marker for studies of mammalian hsp70i gene promoters.

Animals↗
PubMed1997

Expression of the testis-specific HSP70-related gene (hst70 gene) in somatic non-testicular rat tissues revealed by RT-PCR and transgenic mice analysis.

The hst70 gene which belongs to rat HSP70 multigene family is highly expressed in pachytene spermatocytes. Using a transgenic mice model it was found that the promoter of the rat hst70 gene directs the expression of the chloramphenicol acetyl transferase (CAT) reporter gene not only to testis but also to multiple somatic tissues. In wild-type rats the expression of the hst70 gene in tissues other than testis was confirmed by non-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Beside the testis, the CAT expression in transgenic mice and the hst70 gene transcripts in wild-type rats were found in brain, pituitary, epididymis, vas deferens, adrenals, spleen, lung, ovary, oviduct and uterus. The only tissue in which both the CAT expression and the hst70 gene activity has not been found was the liver. These observations suggest possibly more universal, not confined to spermatogenesis, function of the hst70 gene.

Animals↗
PubMed1997

Identification of a microsatellite region composed of a long homopurine/homopyrimidine tract surrounded by AT-rich sequences upstream of the rat stress-inducible hsp 70.1 gene.

A DNA region containing several repetitive motifs has been detected about 1.9 kbp upstream of the transcription unit of the rat stress-inducible hsp 70.1 gene. The most interesting element of this area is a microsatellite sequence (GA)6CAG(TC)24 that consists of an inverted repeat partially overlapping with the long homopurine/homopyrimidine tract (Pu/Py). DNA molecule within the described sequence can theoretically adopt alternate, non-B structures (H-DNA or cruciform) containing single-stranded regions. This microsatellite region is flanked by AT-rich sequences containing several poly(A) tracts. The longest of them with a possible potential to destabilized a double-stranded DNA helix is localized around 160 bp downstream the (GA)6CAG(TC)24. The DNA fragment containing sequences described above was subcloned into the pUC19 vector and the resulting plasmid was subjected to the standard S1 susceptibility assay. Preliminary mapping of the S1 cleavage site indicates for the formation of the non-B-DNA structure within the Pu/Py tract. This is to our knowledge a first report on the existence of a complex microsatellite region on upstream the 5'-end of the hsp 70 gene in mammals.

Animals↗
PubMed1996

Regulation of function of the murine luteinizing hormone receptor promoter by cis- and trans-acting elements in mouse Leydig tumor cells.

The transcriptional activity of various lengths of the 5'-untranslated region (UTR) of the murine LH receptor (R) gene were studied using the luciferase reporter gene in transiently transfected mouse Leydig tumor cells (mLTC-1). Chinese hamster ovarian (CHO) and HeLa cells were used as controls. The basal transcriptional promoter activity in mLTC-1 cells resided within the first 173 base pairs (bp) of the 5'-UTR. Placing an LHR promoter fragment (bases -715/ -56) in front of the Herpes simplex virus thymidine kinase (TK) minimal promoter resulted in a 7-fold increase in luciferase activity. Deletion of bases -56 to -173 of the above construct totally abolished the increased luciferase activity, brought about by the LHR promoter sequences. Basically similar results on LHR promoter function were observed using CHO cells. In contrast, no LHR promoter activity was detected in HeLa cells, indicating a cell specific nature of its function. The first 173 bp promoter domain is GC-rich, with several SP-1 binding domains, and it bound specifically nuclear proteins isolated from mLTC-1 and HeLa cells. RNAse protection assays reconfirmed the presence of several transcription initiation sites within the first 310 bp of the 5'-UTR, also in the absence of the cognate LHR coding sequences. The most distal site at bp -310 did not function in the absence of the first 173 bp of the 5'-UTR. Other transcription initiation sites were identified closer to the translation initiation site. hCG (50 micrograms/l), 8-bromo (Br)-cAMP (100 mumol/l) and cholera toxin (100 microgram/l) displayed qualitatively similar negative effects on the LHR promoter activity in the transfected mLTC-1 cells when the constructs containing at least the first 565 bp of the LHR 5'-UTR were used, but the inhibitory effects were greatly decreased in constructs containing < or = 304 bp of the promoter region. Hence, the hCG/cAMP associated inhibitory effects interact with region(s) located mainly between bp -565 and -305 of the LHR promoter. The inhibitory role of cAMP on LHR gene expression was also confirmed at the level of hCO-binding and hCG stimulated cAMP production of mLTC-1 cells. In conclusion, the current results elucidate the cis- and trans-acting elements in the regulation of expression of the murine LHR gene in cultured mouse Leydig cells. The minimal basal promoter activity is within the first 173 nucleotides of the 5'-UTR and the structural elements of the negative LHR regulation by the cognate hormone and elevated cAMP levels are mainly located within nucleotides -305 to -565 of the 5'-UTR. The function of the murine LHR promoter is similar to, though not identical with that of the rat, but at variance with that of the human LHR gene.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Construction of cDNA library from liver RNA of heat shocked rats and DNA sequence analysis of the clone containing the 3'-end untranslated region (3'UTR) of the heat inducible gene hsp70.2.

cDNA library constructed from liver RNA of rats subjected to hyperthermia was used to isolate divergent 3' end untranslated regions (3'UTR) of heat inducible hsp70.1 and hsp70.2 genes. As a result of a double selection procedure with the use of DNA-DNA hybridization and PCR analysis 9 clones containing cDNA sequences derived from the 3'UTR of the hsp70.2 gene were selected. Nucleotide sequence of the cloned inserts was established and the Northern blot analysis was performed to identify the heat inducible transcript encoded by the hsp70.2 gene.

Animals↗
PubMed1995

A 252 bp upstream region of the rat spermatocyte-specific hst70 gene is sufficient to promote expression of the hst70-CAT hybrid gene in testis and brain of transgenic mice.

The rat hst70 gene belongs to a heat shock hsp70 multigene family and its expression has been detected so far solely in spermatocytes. To investigate the cis-elements responsible for testis-specific expression of the hst70 gene we produced several lines of transgenic mice carrying fragments of the 5'-flanking regions of the hst70 gene fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Hybrid genes of series B were constructed such that, besides the 780 bp, 343 bp and 163 bp 5'-flanking region these plasmids contained no other sequences of the hst70 gene. In hybrid genes of series D the CAT gene was ligated to 343 bp and 252 bp 5'-flanking regions together with the 57 bp of the 5'-end nontranslated (leader) sequences of the hst70 gene. We found that in 780/B, 343/B, 343/D and 252/D adult mice the transgene was specifically and highly expressed in testes. In developing testes the high CAT activity appeared in transgenic mice aged 3 weeks and older. None of the three 163/B transgenic lines exhibited CAT activity in any tissue analyzed. In all CAT expressing lines a weak but significant CAT activity (up to 5% of that in testis) was detected also in the brain. RNase protection assay confirmed that the endogenous hst70 gene transcripts are present in testis as well as in brain of nontransgenic rats and mice. Our data show that the cis-regulatory sequences responsible for testis-specific and developmentally regulated expression of the hst70 gene are localized within the 252 bp region 5' to the gene and neither the 5'-end nor 3'-end nontranslated sequences of the gene are important for this specificity.

Animals↗
PubMed1994

Cloning, nucleotide sequence and expression of rat heat inducible hsp70 gene.

In rat cells hyperthermia induces two hsp70 transcripts of 2.5 kb and 2.7 kb. We have cloned and determined the nucleotide sequence of a gene (named hsp70.1) encoding the 2.5 kb transcript as shown by Northern blot analysis using the 5' end and 3' end specific hybridization probes. It contains an uninterrupted open reading frame of 1926 bp, it encodes a protein of approx. 70,100 Da and the predicted amino acid sequence of its product shows 98% similarity to the mouse hsp70.1 protein. The transcription start site was localized 224 bp upstream the ATG codon by RNase protection and primer extension mapping. Upstream the transcription initiation site several potential regulatory motifs including a TATA box, two Sp1 binding sites, one inverted and one direct CCAAT box and three HSEs (heat shock elements) were found. Transfection experiments with constructs in which the CAT reporter gene was fused to fragments of the 5' end flanking sequences of the isolated gene confirmed that the promoter of the rat hsp70.1 gene is functional and heat inducible.

Albinism↗
PubMed1993

An upstream region of the rat spermatogenesis-specific heat-shock-like Hst70 gene confers testis-specific expression in transgenic mice.

In order to study the temporal and spatial regulation of a rat testis-specific heat-shock-like hst70 gene, an 0.8-kb fragment of its upstream DNA was fused to the lacZ gene and microinjected into one-cell murine embryos. Independent tgHST1 and tgHST2 transgenic mice strains were established, containing about 5-7 and 40-60 transgene copies/haploid genome, respectively. Enzyme assays in various tissues showed that transgene-encoded beta-galactosidase accumulates exclusively in testes of transgenic animals and cannot be detected until 16-17 days after birth. In-situ assays revealed that the enzyme accumulates mainly in pachytene primary spermatocytes. Our data complement previous studies on the endogenous rat hst70 and suggest that its 0.8-kb upstream region contains sufficient information to function as an active spermatogenesis-specific promoter.

Animals↗
PubMed1993

The ERE-like sequence from the promoter region of the testis specific hsp70-related gene is not estrogen responsive.

The 5'flanking region of the hsp70-related gene which is specifically expressed in rat and mouse spermatocytes contains the GGTCAnnnCGACC sequence closely resembling the palindromic consensus ERE (estrogen responsive element, GGTCAnnnTGACC). We used a transient expression assay to study whether this ERE-like sequence can transfer estrogen responsiveness either to heterologous or homologous promoters linked to a reporter CAT gene. We found that the GGTCAnnnCGACC sequence either as an isolated element or in its natural sequence context does not respond to hormone stimulation in MCF-7 and Fe33 cells. This observation points to the importance of the T-nucleotide in a right half-palindrome of ERE for ligand-dependent activation of transcription.

Animals↗