A hypothalamic activator of calmodulin-dependent enzymes is thymosin β4 (1–39)

A new class of stimulators of basal activity of a number of calmodulin-dependent enzymes have been previously isolated from bovine hypothalamus. One of these stimulators, denoted as C₃, has been purified to homogeneity by reverse phase HPLC and tentatively identified as thymosin ₄ (1–39) by mass spectrometry and Edman microsequence analysis. The stimulating effect of C₃ on rabbit skeletal muscle MLCK basal activity was compared with that of thymosin ₁ and thymosin ₄ (16–38). Evidence is presented that all the indicated compounds are Ca²⁺-independent high-affinity MLCK stimulators. The potency of the stimulators in activating the enzyme was: C₃>₄>(CaM+Ca²⁺>₁.This revised version was published online in June 2005 with corrections to the author name Gurvits.     

Yeast mitochondria (Saccharomyces cerevisiae) contain Ca(2+)-independent phospholipase A1 and A2 activities: effect of respiratory state

We demonstrate that both phospholipase A1 and phospholipase A2 are associated with isolated yeast mitochondria (Saccharomyces cerevisiae). Activity assays indicate that, unlike most other mitochondrial phospholipases A, the yeast enzymes are Ca(2+)-independent with acidic (pH 4-5) as well as alkaline (pH 8-9) pH optima. Data obtained with mitochondria isolated from either fermenting or respiring cells, and initial observations with a petite strain, strongly suggest that a phospholipase A2 with an acidic pH optimum functions in the in vivo adaptation and maintenance of mitochondrial membranes required for respiration.     

Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early post-implantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.     

Mouse Chromosome 7

Chair of Committee for Mouse Chromosome 7     

Risk of second primary cancers after Hodgkin's disease by type of treatment: analysis of 2846 patients in the British National Lymphoma Investigation.

OBJECTIVE--To analyse the risk of second primary cancers during long term follow up of patients with Hodgkin''s disease. DESIGN--Cohort study. SETTING--The British National Lymphoma Investigation (a collaborative group of over 60 participating centres in Britain treating lymphomas). PATIENTS--2846 patients first treated for Hodgkin''s disease during 1970-87, for whom follow up was complete in 99.8%. MAIN OUTCOME MEASURES--Second primary cancers; uniform pathology reviews confirmed the diagnosis of Hodgkin''s disease and of second primary non-Hodgkin''s lymphomas. RESULTS--113 second primary cancers occurred. Relative risk of cancer other than Hodgkin''s disease was 2.7 (95% confidence interval 2.3 to 3.3) compared with the general population, with significant risk of leukaemia (16.0(9.1 to 26.0)); non-Hodgkin''s lymphoma (16.8(9.8 to 26.9)); and cancers of the colon (3.2 (1.4 to 6.2)), lung (3.8 (2.6 to 5.4)), bone (15.1 (1.8 to 54.7)), and thyroid (9.4 (1.1 to 33.9)). Absolute excess risk associated with treatment was greater for solid tumours than for leukaemia and lymphomas. Relative risk of leukaemia increased soon after treatment, reaching a peak after five to nine years. It was increased substantially after chemotherapy (27.9 (12.7 to 52.9)), combined treatment with radiotherapy and chemotherapy (21.5 (7.9 to 46.8)), and relative to number of courses of chemotherapy but was not significantly increased after radiotherapy (2.5 (0.1 to 14.1)). Relative risk of non-Hodgkin''s lymphoma increased in the first five years after treatment and remained high but showed no clear relation with type or extent of treatment. Relative risk of solid tumours was less raised initially but increased throughout follow up and for lung cancer 10 years or more after entry was 8.3 (4.0 to 15.3). The risk of solid tumours increased after treatments including radiotherapy and after chemotherapy alone. The risk after chemotherapy increased significantly with time since first treatment. CONCLUSION--The risk of solid cancer, not of leukaemia, is the major long term hazard of treatment for Hodgkin''s disease, and this seemed to apply after chemotherapy as well as after radiotherapy. These risks of second cancers are important in choice of treatment and in follow up of patients, but they are small compared with the great improvements in survival which have been brought about by modern therapeutic methods for Hodgkin''s disease.     

Audit of compliance with antenatal protocols.

OBJECTIVE--To assess the implementation of action protocols dictated by antenatal risk factors noted at the initial (booking) antenatal visit. DESIGN--Retrospective study of 2000 women delivered between 1 March 1990 and 29 March 1991. SETTING--Maternity department of a district general hospital supporting a multiethnic population in inner London. MAIN OUTCOME MEASURES--Comparison of clinical actions performed against those dictated by the department''s protocols. Analysis according to clinical importance, gestation at booking, maternal age, parity, birth order, ethnic origin, and certainty of gestational age. RESULTS--Interobserver agreement between the two auditors was good (kappa statistic for risk factors detected, 0.78; for actions generated, 0.80). Of the 15,658 actions dictated by department protocols, 3673 (23.5%) were actually performed by the clinicians. The 63 combinations of risk factors and actions believed by consultants to be of particular clinical importance had an action rate of 28.3% compared with 18.6% for those considered less important (p < 0.001). Mothers who first visited the hospital antenatal clinic at or before 24 weeks'' gestation had 25.2% of relevant protocols fulfilled (p < 0.001). Compliance was significantly improved in women aged 36 or over (32.4%), black women (24.9%), and cases of uncertain gestation (24.5%). Parity and birth order were not associated with an altered action rate. Ethnic origin deemed as "other" (than white, black, Asian, or oriental) or "unknown" was associated with poor compliance (19.3%). CONCLUSIONS--Compliance to a set of agreed protocols was poor even though a computer system was available and a protocol manual had been distributed. Protocols were more likely to be implemented in women who booked early and in some groups of women deemed at high risk including older mothers, black women, and those denoted as having uncertain gestational age.     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IAl or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P4501Al and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P4501A1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.Some of the data presented here were taken from a thesis submitted by D.K.H. in partial fulfillment of the requirements for the Ph.D. degree in the University of Texas Graduate School of Biomedical Sciences.     

Cytogenetic Definition and Morphogenetic Analysis of Delta, a Gene Affecting Neurogenesis in Drosophila Melanogaster

We have conducted a genetic analysis of a small interval of the third chromosome known to include Delta (Dl), a locus that affects the segregation of the ectoderm into neural and epidermal lineages during embryogenesis and the morphogenesis of some ectodermally derived structures, in Drosophila melanogaster. This analysis has led to the definition of seven independent complementation groups, one of which is Delta, within the interval extending from 91F6-13 to 92A2. Among the extant mutations in these seven loci, only mutations in Dl lead to the so-called neurogenic phenotype: hypertrophy of the nervous system and reduction of the epidermis. Combined cytogenetic and genetic analyses allow us to define absolute proximal (91F5-92A1) and distal (92A2) cytogenetic limits for the Dl locus. We have isolated hypomorphic and amorphic alleles of Dl and find that, for any given allele, there is an inverse correlation between neural hypertrophy and epidermal reduction in embryos and a direct correlation between the severity of embryonic phenotypes in mutant homozygotes and hemizygotes and the imaginal phenotype in heterozygous adults.