Response of murine bone marrow granulocyte-macrophage colony-forming units to hyperthermia in situ

A detailed understanding of how bone marrow stem cell progenitors are affected by heat is prerequisite to predicting how whole-body or regional hyperthermia protocols may affect bone marrow function. This investigation reports the reproductive integrity of murine tibial bone marrow granulocyte-macrophage colony-forming units (CFU-GM) after in situ hyperthermia. Heat was applied by water bath immersion of the leg of male BALB/c mice anesthetized with 90 mg/kg pentobarbital given subcutaneously. Tibial and rectal temperatures were monitored in representative animals by microthermocouples (tip diameter approximately 100 microns). By approximately 3 min after immersion of the limb, marrow temperature was within 0.3 degree C of water bath temperature (O'Hara et al., Int. J. Hyperthermia 5, 589-601, 1989) and was within 0.1 degree C by 5 min after immersion. The CFU-GM were cultured in "lung-conditioned" McCoy's 5A medium supplemented with 15% fetal calf serum and 0.3% Bacto agar. In situ heating of tibial marrow to exposure temperatures of 42, 42.5, 43, 44, and 45 degrees C gave D0's (+/- 95% CI) of 91 +/- 44, 44 +/- 27, 27 +/- 2.2, 16 +/- 6, and 7 +/- 4 min, respectively. Heating to 41.5 degrees C for up to 180 min did not result in cytotoxicity. Development of thermotolerance after approximately 100 min of heating was apparent by the presence of a "resistant tail" of the 42 degrees C survival curve. A plot of D0 vs water bath temperature was bimodal with an inflection point at approximately 42.5 degrees C. The inactivation enthalpy for temperatures above 42.5 degrees C was 586 kJ/mol (140 kcal/mol) and for temperatures below 42.5 degrees C was estimated to be 1205 kJ/mol (288 kcal/mol). These results show that CFU-GM can be heated predictably in situ, can be inactivated with thermal exposures as low as 42 degrees C, and are capable of developing thermotolerance. These findings underscore the necessity to understand stem cell inactivation by hyperthermia in situ prior to widespread implementation of clinical hyperthermia protocols where bone marrow may be included in the treatment field.     

The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling.     

Eicosanoids and their role in immune modulation in fish—a brief overview

Eicosanoids have been demonstrated to play a central role in immune regulation in mammals brought about by their direct effects on cells such as macrophages and lymphocytes or by their indirect effects via cytokines. Studies have shown that fish mononuclear phagocytes, granulocytes and thrombocytes synthesize and release both cyclooxygenase- and lipoxygenase-derived products such as prostaglandin E₂, leukotriene B₄ and lipoxin A₄. Whether lymphocytes have the ability to generate leukotrienes and lipoxins is still unclear but they do appear to have 12-lipoxygenase activity that leads to the generation of 12-hydroxy fatty acid derivatives. As in mammals, leukotriene and lipoxin biosynthesis requires the presence of a 5-lipoxygenase activating protein-like molecule that is sensitive to the action of the specific inhibitor, MK-886. The prostaglandin-generating ability of trout macrophages can be altered by incubation with lipopolysaccharide suggesting the possible presence of an inducible cyclooxygenase activity. Prostaglandins have been found to suppress the mitogen-induced proliferation of trout leucocytes and the generation of humoral antibody and plasma cells both in vivo and in vitro. The lipoxygenase products, leukotriene B₄ and lipoxin A₄ have more variable effects ranging from inhibition to stimulation depending on the assay system employed. Overall, there is clear evidence that eicosanoids play a role in immune regulation in fish in a similar way to that reported in mammals.     

Genetic diversity in and phylogenetic relationships of the Brazilian endemic cotton,Gossypium mustelinum (Malvaceae)

Gossypium mustelinum, one of five tetraploid species in the cotton genus, is geographically restricted to a few states in NE Brazil. Allozyme analysis was used to assess levels and patterns of genetic diversity inG. mustelinum and its relationship to the other tetraploid species. Genetic variation was low, with only 6 of 50 loci examined being polymorphic, a mean of 1.14 alleles per locus and a mean panmictic heterozygosity of 0.08. These estimates are low relative to other tetraploid cotton species, but are typical of island endemics. Interpopulational genetic identities were uniformly high, lending support to the concept of there being only one wild species of Brazilian cotton. The limited allelic diversity observed was correlated with geographical distribution, although variability is so limited in the species that geographically marginal populations are electrophoretically ordinary. Phylogenetic and phenetic analyses demonstrate thatG. mustelinum is isolated among polyploid cotton species, occupying one of the three basal clades resulting from an early radiation of polyploid taxa subsequent to polyploid formation. We suggest thatG. mustelinum represents a paleoendemic that presently exists as a series of widely scattered, relictual populations. Despite several centuries of sympatric cultivation ofG. barbadense andG. hirsutum, there was little evidence of interspecific introgression of alleles from cultivated cottons intoG. mustelinum.     

Binding-protein expression is subject to temporal,developmental and stress-induced regulation in terminally differentiated soybean organs

Binding protein (BiP) is a widely distributed and highly conserved endoplasmic-reticulum luminal protein that has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of misfolded polypeptides. Analysis of cDNA sequences and genomic blots indicates that soybeans (Glycine max L. Merr.) possess a small gene family encoding BiP. The deduced sequence of BiP is very similar to that of other plant BiPs. We have examined the expression of BiP in several different terminally differentiated soybean organs including leaves, pods and seed cotyledons. Expression of BiP mRNA increases during leaf expansion while levels of BiP protein decrease. Leaf BiP mRNA is subject to temporal control, exhibiting a large difference in expression in a few hours between dusk and night. The expression of BiP mRNA varies in direct correlation with accumulation of seed storage proteins. The hybridization suggests that maturing-seed BiP is likely to be a different isoform from vegetative BiPs. Levels of BiP protein in maturing seeds vary with BiP mRNA. High levels of BiP mRNA are detected after 3 d of seedling growth. Little change in either BiP mRNA or protein levels was detected in maturing soybean pods, although BiP-protein levels decrease in fully mature pods. Persistent wounding of leaves by whiteflies induces massive overexpression of BiP mRNA while only slightly increasing BiP-protein levels. In contrast single-event puncture wounding only slightly induces additional BiP expression above the temporal variations. These observations indicate that BiP is not constitutively expressed in terminally differentiated plant organs. Expression of BiP is highest during the developmental stages of leaves, pods and seeds when their constituent cells are producing seed or vegetative storage proteins, and appears to be subject to complex regulation, including developmental, temporal and wounding.The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.Abbreviations BiP binding protein The sequences reported in this paper have been submitted to Gen-Bank and are identified with the accession numbers BiP-A (U08384), BiP-B (U08383), BiP-C (U08382) and -1,3 glucanase (U08405).     

Home care for patients with suspected myocardial infarction: use made by general practitioners of a hospital team for initial management.

Two hundred and sixty three general practitioners were offered the use of a hospital based service consisting of a medical senior house officer, a nurse attached to a coronary care unit, and a specially equipped ambulance estate car to help with the initial management of patients with suspected myocardial infarction who might be suitable for home care. One hundred and sixty nine general practitioners registered as potential users of this service; during 22 months they called the hospital team to see 271 patients, 235 of whom the team suspected had indeed suffered a myocardial infarction. During the same period, however, these general practitioners also admitted 317 patients with suspected myocardial infarction directly to hospital. Other general practitioners admitted 323 patients and deputising doctors 258. A further 529 patients with suspected infarction were admitted without the intervention of a general practitioner. Of the patients seen by the team, 54 required immediate admission to hospital; 17 of the remaining patients who initially appeared suitable for home care later required admission to hospital. In a large city such as Nottingham the provision of hospital based facilities to help general practitioners with home management is unlikely to make an appreciable impact on the overall pattern of care of patients with suspected myocardial infarction.     

Screening and genetic counseling for beta-thalassemia trait in a population unselected for interest: effects on knowledge and mood.

To evaluate the effects of genetic screening and counseling in a population unselected for interest, adults in a health maintenance organization (HMO) were screened for beta-thalassemia trait as part of health care or multiphasic screening. Counseling was provided by either a trained physician or a videotape containing the same information, followed by an opportunity to question a trained physician. Knowledge of thalassemia, knowledge of genetics, and mood were assessed by standardized questionnaires and by interview immediately before and after counseling. Compared to controls, trait subjects demonstrated significant learning about thalassemia (P less than .001) and about genetics (P less than .001) and recorded significant mood changes, namely, surprise (startle) (P less than .05), increased alertness (decreased deactivation) (P less than .05), and decreased skepticism (P less than .01). Screening and genetic counseling for beta-thalassemia trait conducted as part of multiphasic screening of the population of a HMO, essentially and unselected population, can result in significant overall learning with acceptable effects on mood.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

A Burkitt-lymphoma variant translocation (2p-; 8q+) in a patient with ALL,L3 (Burkitt type)

Summary A patient with acute lymphoblastic leukemia (ALL) whose cells had L3 (Burkitt-type) morphology was found to have a variant translocation t(2;8)(p13;q24), that has been reported only in Burkitt lymphomas. This observation provides additional evidence for a close association of particular karyotypic abnormalities with both Burkitt-type ALL and Burkitt lymphoma.     

Cryogenic gamma irradiation of prototype pork and chicken and antagonistic effect between Clostridium botulinum types A and B.

Inoculated, irradiated pork (2,300 cans) and chicken (2,000 cans) pack studies were performed to establish the 12D dose for these foods. Each can was inoculated with a mixture of 10(6) spores of each of 10 strains of Clostridium botulinum (five type A and five type B), or a total of 10(7) spores. The cans received a series of increasing doses of gamma rays (60Co) at -30 +/- 10 degrees C; they were incubated for 6 months at 30 +/- 2 degrees C and examined for swelling, toxicity, and recoverable botulinal cells. The highest rate of swelling for both foods occurred within the first week of incubation, and maximum swelling was observed within 4 to 5 weeks. The minimal experimental sterilizing dose (ESD) based on flat, nontoxic sterile cans was 3.0 less than ESD less than or equal to 3.2 Mrad for pork and 4.0 less than ESD less than or equal to 4.2 Mrad for chicken. An analysis of the partial spoilage data by extreme-value statistics indicated with 90% confidence that the rate of spore death in the two foods was not a normal distribution, but appeared to favor a shifted exponential function. Based on the latter distribution, and assuming one most resistant strain in the mixture of 10 used, the 12D dose computed to 4.37 Mrad, with a shoulder of 0.11 Mrad, for pork and to 4.27 Mrad, with a shoulder of 0.51 Mrad, for chicken. An assumption that there were two or more most resistant strains in the inoculum progressively lowered the 12D dose. There was an apparent antagonism between the irradiated type A and B viable strains in the two foods. Cans with type B cells and toxin predominated over cans with type A cells and toxin, but cans with a mixture of type A and B toxins predominated over cans with a mixture of Type A and B cells. At the highest sublethal doses, only type A cells survived in pork, but in chicken there was a least one type B strain that was at least as resistant as type A strains.