Correlation of heat resistance and HSP-70A mRNA levels in human tumor cells measured by competitive quantitative polymerase chain reaction

PURPOSE: Several HSP-70 genes have been cloned and sequenced in human cells. Among these genes, the HSP-70A mRNA and protein levels correlate best with the development and decay of thermotolerance and intrinsic thermal sensitivity. Leukemic and nonleukemic human tumor cells express low levels of the normally heat inducible HSP-70A mRNA in control nonheated cells. Using a competitive quantitative polymerase chain reaction, we have measured the levels of mRNA for this gene and have correlated it with both transient and intrinsic thermal sensitivity of tumor cells. Such studies were also extended to tumor samples obtained from patients. METHODS AND MATERIALS: In these studies, the plasmid phHSP-70 which contains the entire human HSP-70A gene was modified by the insertion of the T7 promoter at the 5'-end untranslated region as well as the insertion of a 23 bp synthetic linker at the BamH1 site in the promoter region of the HSP-70A gene. The PCR primers were located such that the amplified fragment contained the linker. Using the T7 polymerase, the HSP-70A mRNA was transcribed from this plasmid (phHSP-70L) in vitro. A known amount of HSP-70A mRNA was then added to the total RNA prepared from the cell samples or from the tumor tissues obtained from patients. Using the components of the PCR reaction plus known amounts of HSP-70A mRNA synthesized from phHSP-70L and unknown amounts of total cellular RNA, the samples were amplified and analysed on a denaturing acrylamide gel. The PCR products obtained from phHSP-70L were 23 bp larger than the PCR products obtained from the cell samples due to the addition of the synthetic linker to the HSP-70A gene in phHSP-70L and therefore, the two products could be easily distinguished from each other and quantitated. The alpha-32P-dCTP incorporated in each sample was quantitated by AMBIS Scanner. When the 32P-counts were equal in the known and the unknown samples, the amount of the HSP-70A mRNA was taken to be equal in the known and the unknown sample. RESULTS: The results show that HSP-70A mRNA levels can be used to predict the survival levels during the development and decay of thermotolerance. In nonleukemic human tumor cell lines, there are as much as 40-50-fold induction of HSP-70A mRNA levels during the peak of thermotolerance. In leukemic cell lines, however, HSP-70A mRNA levels are induced only by three-fold during the same time period. These differences between the levels of HSP-70A mRNA positively correlate with the amount of tolerance development in leukemic and nonleukemic tumor cells. HSP-70A mRNA levels also vary in different tumor cells under nonheated conditions and there is a positive correlation between HSP-70A mRNA levels in nonleukemic human tumor cells and the level of their intrinsic thermal resistance. CONCLUSION: HSP-70A mRNA levels can be used to predict the intrinsic thermal sensitivity of nonleukemic human tumor cells.     

The metabolism of 3-deoxy-3-fluoro-D-glucose by Locusta migratoria and Schistocerca gregaria

3-Deoxy-3-fluoro-D-glucose (3FG) administered by injection is toxic to adult Locusta migratoria or Schistocerca gregaria (LD50, 4.8 mg/g). temperature-programmed and isothermal gas chromatographic analysis of poisoned locust haemolymph reveals the presence of a fluorinated metabolite identified as 3-deoxy-3-fluoro-D-glucitol (3FGL). The enzymes responsible for the accumulation of this metabolite are located in the fat body of the insect and partially purified as aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) and sorbitol dehydrogenase (L-iditol: NAD+ 5-oxidoreductase EC 1.1.1.14) 3FGL is shown to be both a competitive inhibitor of the NAD-linked sorbitol dehydrogenase with Ki 8?x 10(-2) M as well as a substrate with Km 0.5 M. A kinetic rate equation is derived and verified to account for the kinetic duality of 3FGL. These results partially explain the toxic effects of 3FG and are consistent with the presence of a hitherto undetected sorbitol metabolism in locusts.     

In-situ emergency paediatric surgery in the intensive care unit

The role of surgery in the intensive care unit (ICU) remains unclear. Although previously shown not to increase morbidity for patent ductus arteriosus ligation, Broviac catheter insertion, and recently, general neonatal and paediatric surgery, there remains a reluctance to operate on sick patients in the ICU (in-situ surgery, ISS). A retrospective study of 25 critically ill children and neonates who underwent ISS was performed. Surgery was aided by operating loupes and a high-intensity headlight. ISS was not associated with any morbidity, and although a 36% mortality occurred in this small series, in no case was this due to ISS. ISS avoids the risks of transfer to the operating theatre and the potential delays in theatre access. Our results suggest that ISS in a tertiary-level paediatric surgical hospital is safe and does not impact adversely on clinical outcome.     

Structural organization of 2C-terminal region and its analogs in a subunit of ribonucleotide reductase from herpes virus

By means of conformational analysis, the spatial structure and conformational potential of the H-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-OH molecule, which corresponds to sequence 329-337 of the subunit 2 C-terminal region of the herpes virus ribonucleotide reductase, were studied. It was shown that its spatial organization can be described by a set of 17 low-energy conformations of the backbone. The "reverse conformational problem" for this molecule was solved to enable the prediction of a series of synthetic analogues matching the set of low-energy, potentially physiologically active conformations.     

The pharmacokinetics and metabolism of heptapeptide--a prospective synthetic analog of tuftsin with psychostimulating action in rats

The lymphocyte blast transformation test (LBTT) with three tuberculin dilutions was used to examine 190 patients with varying pulmonary tuberculosis activity, of them 63 patients received chemotherapy. According to the blast formation in the patients' cultured peripheral blood cells by three tuberculin dilutions, a correlation was found between the clinical manifestations of the process and the functional activity of T lymphocytes. Thus, the greatest percentage (500 TU) of blasts in LBTT per mean PPD dose was detectable in patients with low LBTT results by three tuberculin dilutions with positive dynamics during chemotherapy. With further positive dynamics, the proportion of blasts in the cultured peripheral blood cells was highest per high PPD doses (5000 TU). On the contrary, patients with progressive tuberculosis displayed a oppositely directed phasic pattern.     

Monoclonal endothelial cells in appetite suppressant-associated pulmonary hypertension

Anorexigens such as aminorex fumarate and dexfenfluramine are associated with the development of severe pulmonary hypertension (PH), which clinically and histopathologically is considered indistinguishable from idiopathic or primary pulmonary hypertension (PPH). For the current study, we asked whether anorexigen-associated PH is characterized by monoclonal pulmonary endothelial cell proliferation (such as in PPH) or, alternatively, is associated with a polyclonal endothelial cell proliferation as found in secondary PH. Analysis of clonality by the human androgen receptor assay was performed in microdissected endothelial cells of plexiform lesions of two patients with anorexigen-associated PH. The four plexiform lesions of Patient 1 and the six of Patient 2 with anorexigen-associated PH exhibited a monoclonal expansion of pulmonary endothelial cells, with a mean clonality ratio of 0.03 +/- 0.01 SE. Our results indicate that appetite suppressant-associated PH is identical to PPH not only in clinical and histopathologic features but also, at a molecular level, in terms of the monoclonal nature of the endothelial cell proliferation. The anorexigens may accelerate the growth of pulmonary endothelial cells in patients with predisposition to develop PPH.     

Expression and possible role of muscle-type carnitine palmitoyltransferase I during sperm development in the rat

Because we had found whole testis from adult rats to be much richer in the messenger RNA for the muscle (M) than for the liver (L) form of mitochondrial carnitine palmitoyltransferase I (CPT I), we sought to determine which cell type(s) accounts for this expression pattern and how it might relate to reproductive function. Studies with immature (14-day-old) and adult animals included 1) Northern blot analysis of testis mRNA; 2) in situ hybridization with slices of testis; 3) enzyme assays for CPT I, CPT II, and carnitine acetyltransferase (CAT) in testicular germ cells and nongerm cells, together with measurement of the malonyl-coenzyme A (CoA) sensitivity and affinity for carnitine of CPT I; 4) labeling of testicular CPT I with [3H]etomoxir, a covalent inhibitor of the enzyme; and 5) the response of testicular and nontesticular CPT I to dietary etomoxir. The data established the following: 1) L-CPT I was the sole isoform detected in immature testis. 2) Expression of the M-CPT I gene was associated only with meiotic and postmeiotic germ cells. 3) Adult testis contains a mixture of the L- and M-CPT I enzymes, the L and M form dominating in extratubular cells and spermatids, respectively. Mature epididymal spermatozoa appear to be devoid of CPT I activity while possessing abundant levels of CPT II and CAT. 4) Five days of dietary etomoxir treatment at a dose that resulted in essentially complete inhibition of CPT I in liver, heart, skeletal muscle, and kidney was totally without effect on either the L- or M-type enzyme in the testis of mature rats. The data point to an important role for transient expression of M-CPT I, coupled with sustained activity of CAT, in the maturation and/or function of rat sperm. They also suggest that, at least in the case of one CPT I inhibitor (etomoxir), the testis is unusually resistant to the agent when given orally.     

Conjugation of classic Bowman-Birk soy inhibitor with a copolymer of ethylene oxide and propylene oxide

A classical soybean inhibitor of the Bowman-Birk type (BBI) with a copolymer of ethylene oxide and propylene oxide (PE) has been synthesized. The BBI-PE conjugate contain five covalently bound polymeric chains per one protein molecule and retains its capacity to inhibit trypsin (Ki = 10(-10) M), alpha-chymotrypsin (Ki = 7 x 10(-8) M) and human granulocyte elastase (Ki = 3 x 10(-8) M). The preservation of the antiproteinase activity in the antichymotrypsin center creates a prerequisite for the manifestation of the anticarcinogenic effect of the inhibitor.