Operational feasibility of routine bacterial monitoring of platelets.

Bacterial contamination of platelets poses the greatest risk of mortality and morbidity to platelet transfusion recipients. Some European countries have introduced routine bacterial monitoring of platelets to reduce the risk of transmission of bacteria. A pilot study was carried out at the Northern Ireland Blood Transfusion Service, using the BacT/ALERT automated culture system, to assess the operational feasibility of routine bacterial monitoring of platelets. About 4885 platelet concentrates (PCs) were tested in a 1-year period. Of the 28 (0.57%) initial reactive cultures, 13 (46%) were reproducible on repeat culturing. Of these, 10 were detected within 24 h of incubation either in aerobic or both aerobic and anaerobic culture bottles. A sample of time-expired units (423) that had initial negative culture results remained negative when retested on day 8. About 213 time-expired units were subjected to routine quality assessment and more than 85% were found to conform to quality standards specified in the UKBTS guidelines for platelet count (> or =240 x 10(9) per adult dose PC) and pH (6.4-7.4). There was a reduction in the platelet count because of the volume removed (15 mL) for sampling. Routine bacterial testing with day 2 sampling and a negative culture result after 24 h as a mandatory release criterion would improve product safety. Implementation of 100% testing would be operationally feasible but may require extension of the shelf life if unacceptable wastage is to be avoided.

Duchenne muscular dystrophy--parental perceptions.

Quality of life and availability of services are important for boys with Duchenne muscular dystrophy (DMD) and their families. Families attending our neuromuscular clinic completed a questionnaire on parental perception regarding the importance of services, health issues, and quality of life issues both "now" and "in the future." Eighty-nine percent of the families (31/35) completed questionnaires. Services and health issues related to prolonging ambulation were most important, especially for the parents of younger boys. Mental health issues such as social isolation, anger, and depression were very important, particularly for the families of older boys and were anticipated to be more important in the future. Pediatricians should be aware of both the immediate needs of families to meet the physical and emotional challenges of DMD and the increasing requirement to address the social needs of these patients and their families as the boys become older.

Hypersensitivity to DNA damage leads to increased apoptosis during early mouse development.

Gastrulation in mice is associated with the start of extreme proliferation and differentiation. The potential cost to the embryo of a very rapid proliferation rate is a high production of damaged cells. We demonstrate a novel surveillance mechanism for the elimination of cells damaged by ionizing radiation during mouse gastrulation. During this restricted developmental window, the embryo becomes hypersensitive to DNA damage induced by low dose irradiation (<0.5 Gy) and undergoes apoptosis without cell cycle arrest. Intriguingly, embryonic cells, including germ cell progenitors, but not extraembryonic cells, become hypersensitive to genotoxic stress and undergo Atm- and p53-dependent apoptosis. Thus, hypersensitivity to apoptosis in the early mouse embryo is a cell fate-dependent mechanism to ensure genomic integrity during a period of extreme proliferation and differentiation.

Reprogramming the cell cycle for endoreduplication in rodent trophoblast cells.

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34(cdk1) complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

Extracellular matrix remodeling as a regulator of stromal-epithelial interactions during mammary gland development, involution and carcinogenesis.

An intact basement membrane is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or remodeling of the basement membrane occurs during normal development as well as in the disease state. Stromelysin-1 (SL-1), a member of the matrix metalloproteinase (MMP) family, was one of the first proteinases found to be associated with cancer. In this review we describe the role of MMPs in normal mammary gland involution. To examine the importance of basement membrane during development in vivo, we altered the MMP and tissue inhibitor of metalloproteinases (TIMP) balance in mammary gland. Inhibition of MMP synthesis by glucocorticoids or implants or transgenic overexpression of TIMP-1 delays matrix degradation and the involution process after weaning. The mammary glands from transgenic mice that inappropriately express autoactivating isoforms of SL-1 are both functionally and morphologically altered throughout development. Transgenic mammary glands have supernumerary branches, and show precocious development of alveoli that express beta-casein expression and undergo unscheduled apoptosis during pregnancy. This is accompanied by progressive development of an altered stroma, which resembles that of a wound site or a tumor, and becomes fibrotic after postweaning involution, and by development of neoplasias. These data suggest that MMPs and disruption of the basement membrane may play key roles in branching morphogenesis of mammary gland, apoptosis, and stromal fibrosis as well as in induction and progression of breast cancer. These observations suggest that SL-1 and other MMPs may be useful targets for therapeutic intervention in cancer.

Extracellular matrix remodeling and the regulation of epithelial-stromal interactions during differentiation and involution.

An intact basement membrane is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or remodeling of the basement membrane occurs during normal development as well as in the disease state. To examine the importance of basement membrane during development in vivo, we altered the matrix metalloproteinase and tissue inhibitor of metalloproteinases balance in mammary gland. Inhibition of matrix metalloproteinase synthesis by glucocorticoids or implants or transgenic overexpression of tissue inhibitor of metalloproteinases -1 delays matrix degradation and the involution process after weaning. The mammary glands from transgenic mice that inappropriately express auto-activating isoforms of stromelysin-1 are both functionally and morphologically altered throughout development. Transgenic mammary glands have supernumerary branches, and show precocious development of alveoli that express beta-casein expression and undergo unscheduled apoptosis during pregnancy. This is accompanied by progressive development of an altered stroma, which becomes fibrotic after postweaning involution, and by development of neoplasias. These data suggest that metalloproteinases and disruption of the basement membrane may play key roles in branching morphogenesis of mammary gland, cell cycle, apoptosis, and stromal fibrosis as well as in induction and progression of breast cancer.

Mohawk English in the medical interview.

Mohawk English uses metalinguistic cues that reflect Iroquoian grammatical and sociolinguistic patterning. Although phrases like "It seems to me" are common in many varieties of English, Mohawk English speakers use these words and phrases in subtly different ways from other speakers of English. These differences could lead to misinterpretation by health care providers.

Phosphorylation of Src mutants at Tyr 527 in fibroblasts does not correlate with in vitro phosphorylation by CSK.

In normal fibroblasts, the product of the cellular src gene, p60c-src or Src, is repressed by phosphorylation at its C-terminal tyrosine residue, Tyr 527. Mutations in Src that prevent phosphorylation cause enzymatic activation and malignant transformation. The tyrosine kinases that phosphorylate Src at Tyr 527 in vivo have not been identified, but a tyrosine kinase known as CSK is an excellent candidate. CSK has the unusual ability to phosphorylate Src in vitro only at Tyr 527. To examine whether CSK has the appropriate sequence specificy to explain the phosphorylation of Src at Tyr 527 in fibroblasts, we have made use of a set of C-terminal substitution mutants of Src. These mutants were previously characterized for their levels of Tyr 527 phosphorylation when expressed in Rat2 fibroblasts. The ability of CSK to phosphorylate selected mutants has now been tested, using both in vitro phosphorylation assays and co-expression of CSK with the Src mutants in a heterologous organism, Saccharomyces cerevisiae. We also tested whether the mutant Src molecules could autophosphorylate at Try 527, by examining the phosphorylation state of catalytically active forms expressed in the absence of CSK in yeast cells. The results show that CSK has strict sequence specificity for the normal Src sequence, although it can also phosphorylate the Lck sequence. The other mutant Src molecules tested were not phophorylated by CSK, even though some of these mutants are highly phosphorylated at Tyr 527 in Rat 2 cells. All the mutants that are phosphorylated at Tyr 527 in Rat2 cells are also able to autophosphorylate at Tyr 527. The results suggest that CSK, autophosphorylation, and phosphorylation by kinases other than CSK, may all contribution to repressing Src catalytic activity in fibroblasts.

Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src.

The protein-tyrosine kinase activity of the proto-oncogene product p60c-src is negatively regulated by the phosphorylation of a tyrosine residue close to the C terminus, tyrosine 527. The phosphorylation might be catalysed by a so-far-unidentified tyrosine kinase, distinct from p60c-src. Recently we purified a protein-tyrosine kinase that specifically phosphorylates tyrosine 527 of p60c-src from neonatal rat brain. We have now confirmed the specificity of this enzyme by using a mutant p60c-src that has a phenylalanine instead of tyrosine 527, and cloned a complementary DNA that encodes the enzyme. The enzyme is similar to kinases of the src family in that it has two conserved regions, Src-homology regions 2 and 3, upstream of a tyrosine kinase domain. The amino-acid identity of each region is no more than 47%, however, and the enzyme lacks phosphorylation sites corresponding to tyrosines 416 and 527 of p60c-src and has no myristylation signal. These results suggest that this protein-tyrosine kinase, which might negatively regulate p60c-src, represents a new type of tyrosine kinase.

Properties of tripartite chimeras between Src and Lck.

To evaluate the possible regulatory effects of interactions between different domains of Src-family kinases, we have assembled five chimeric molecules containing parts of p60c-src (Src) and p56lck (Lck). Chimeras contained the N-terminal portion, kinase domain or C-terminal tail from either of the parent molecules. None of the four full-length hybrid proteins induced morphological transformation of NIH3T3 cells, nor stimulated phosphorylation of cell proteins on tyrosine residues, suggesting that their protein-tyrosine kinase activities were repressed appropriately in fibroblastic cells. However, two hybrid proteins, SLS and SLL, containing the Src N-terminal region, Lck kinase domain, and either Src or Lck tail, respectively, inefficiently induced anchorage-independent growth. Sites of phosphorylation in these molecules were determined by two-dimensional peptide mapping. SLS and SLL were both phosphorylated at their C-terminal tyrosine residues similarly to the parental molecules. Curiously, both proteins were also significantly phosphorylated at tyrosine 416, a feature of transforming variants of Src and Lck. A modified hybrid, SLO, containing the N-terminal region of Src, kinase domain of Lck and a truncated C-terminus, fully transformed cells and stimulated phosphorylation of cell proteins at tyrosine. Comparison of SLS, SLL and SLO suggests that the full-length hybrids are repressed by their C-terminal phosphorylated tyrosine residues in vivo. Consistent with this, SLO resembled activated Src in being cytoskeletal, and SLS and SLL resembled nontransforming Src and were not cytoskeletal. Hybrids with an Lck N-terminal region were cytoskeletal, like Lck itself, even though they were not transforming, suggesting that cytoskeletal localization of Lck is determined through its N terminus. Curiously, the hybrid molecules appeared not to be regulated in vitro. The specific activities of SLS, SLL and Src were approximately equal, but the specific activity of SLO was not increased, being much less than that of activated Src. Enzymatic dephosphorylation stimulated the in vitro kinase activity of parental Src, but not of SLS or SLL. These observations suggest that chimeric Src-Lck molecules are regulated in the cell, but not in vitro.

Acidic residues at the carboxyl terminus of p60c-src are required for regulation of tyrosine kinase activity and transformation.

Protein phosphorylation sites act to transduce signals into changes in enzymatic activity, representing a point of interaction within a regulatory pathway. The amino acid sequence surrounding a phosphorylation site may well have several functions, including recognition by an appropriate kinase. By generating random mutations in its immediate vicinity, we have examined the sequence requirements of a regulatory tyrosine phosphorylation site, Tyr527, in the proto-oncogene product, p60c-src. The transforming and kinase activities of p60c-src are repressed by phosphorylation of Tyr527. Mutations were made around Tyr527 without changing Tyr527 or the kinase domain. Twenty-nine mutants were sequenced and classified as transforming or nontransforming for Rat-2 cells. Nontransforming mutants contained a surprising variety of COOH-terminal mutations, although acidic residues were present at positions 518 and 524 in all nontransforming mutants. Transforming mutants that contained single-residue changes at Asp518 and Ser522 demonstrated the importance of these residues. Other transforming mutants contained two or more substitutions, but the results are most simply explained if residues Glu524 and Thr523 are also important for normal regulation. Transforming mutations reduced the phosphorylation of Tyr527. We conclude that only a few of the residues in the COOH terminus other than Tyr527 are required to ensure normal phosphorylation and repression of activity in fibroblasts. Other residues may have been conserved during evolution to permit normal function and regulation in other cell types.

Structural differences between repressed and derepressed forms of p60c-src.

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

Cooperative transforming activities of ras, myc, and src viral oncogenes in nonestablished rat adrenocortical cells.

Early-passage rat adrenocortical cells were infected with Kirsten murine sarcoma virus and MMCV mouse myc virus, two retroviruses carrying the v-Ki-ras and v-myc oncogenes, respectively. Efficient morphological transformation required coinfection with the two viruses, was dependent on the presence of high serum concentrations, and was not immediately accompanied by growth in soft agar. The doubly infected cells coordinately acquired the capacity for anchorage- and serum-independent growth during passage in culture. The appearance of such highly transformed cells was correlated with the emergence of a dominant clone, as suggested by an analysis of retrovirus integration sites. These results indicate that the concerted expression of v-Ki-ras and v-myc could induce rapid morphological transformation of nonestablished adrenocortical cells but that an additional genetic or epigenetic event was required to permit full transformation by these two oncogenes. In contrast, v-src, introduced by retrovirus infection in conjunction with v-myc, rapidly induced serum- and anchorage-independent growth. Therefore, the p60v-src protein-tyrosine kinase, unlike p21v-ras, is apparently not restricted in the induction of a highly transformed phenotype in adrenocortical cells. This system provides an in vitro model for the progressive transformation of epithelial cells by dominantly acting oncogenes.

p56lck protein-tyrosine kinase is cytoskeletal and does not bind to polyomavirus middle T antigen.

p56lck and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56lck from introduced DNA molecules to compare the subcellular localizations of p60c-src and p56lck and their abilities to bind polyomavirus middle T antigen (mT). p56lck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60c-src is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60c-src binds to mT, but p56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60c-src mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60c-src to bind mT and be soluble, we tested whether the different localization and mT binding properties of p56lck and p60c-src were dictated by their different carboxy termini. A protein consisting largely of p60c-src sequences but carrying a p56lck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60c-src not only require sequences common to the carboxy termini of p60c-src and p56lck, but also require sequences unique to the body of p60c-src.

The carboxy-terminal sequence of p56lck can regulate p60c-src.

A chimera containing the coding region for residues 1 to 516 of p60c-src and residues 495 to 509 (the carboxy terminus) of p56lck was constructed and expressed in mouse fibroblasts. The chimeric protein appeared to be phosphorylated and regulated in the same fashion as p60c-src.

Potential positive and negative autoregulation of p60c-src by intermolecular autophosphorylation.

The product of the protooncogene c-src is a protein-tyrosine kinase, p60c-src, that is normally inhibited by phosphorylation at a tyrosine residue close to the C terminus (Tyr-527). If activated by dephosphorylation of Tyr-527, or by other means, p60c-src becomes phosphorylated at a tyrosine residue in the catalytic domain (Tyr-416). To test whether either or both of these tyrosines can be phosphorylated by p60c-src itself, we have created four mutations in c-src. One mutant product can receive but cannot donate phosphate, and other mutants are capable of catalysis but lack phosphorylation sites. The mutant genes were expressed singly or in combination in yeast. Analysis of the phosphorylation of mutant p60c-src in the yeast cells and in immunoprecipitates showed that p60c-src molecules can phosphorylate each other at Tyr-416 and -527. Prohibiting intramolecular phosphorylation had little effect on reaction rates and extents, suggesting that intermolecular phosphorylation predominates. If the same situation pertains in the milieu of the vertebrate fibroblast, phosphorylation of one p60c-src by another at Tyr-416 or -527 could permit positive or negative autoregulation.

Expression of viral p21ras during acquisition of a transformed phenotype by rat adrenal cortex cells infected with Kirsten murine sarcoma virus.

Rat adrenal cortex cells infected with Kirsten murine sarcoma virus acquire a transformed phenotype in a progressive fashion. The expression of the viral p21ras does not appear to correlate with the degree of transformation of the adrenocortical cells but rather is produced at similar levels as the culture becomes transformed. This indicates that the expression of an oncogenic form of p21ras is not of itself sufficient to completely transform rat adrenal cortex cells.