A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast.

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.

Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization.

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.

Two related kinesins, klp5+ and klp6+, foster microtubule disassembly and are required for meiosis in fission yeast.

The kinesin superfamily of microtubule motor proteins is important in many cellular processes, including mitosis and meiosis, vesicle transport, and the establishment and maintenance of cell polarity. We have characterized two related kinesins in fission yeast, klp5+ and klp6+,, that are amino-terminal motors of the KIP3 subfamily. Analysis of null mutants demonstrates that neither klp5+ nor klp6+, individually or together, is essential for vegetative growth, although these mutants have altered microtubule behavior. klp5Delta and klp6Delta are resistant to high concentrations of the microtubule poison thiabendazole and have abnormally long cytoplasmic microtubules that can curl around the ends of the cell. This phenotype is greatly enhanced in the cell cycle mutant cdc25-22, leading to a bent, asymmetric cell morphology as cells elongate during cell cycle arrest. Klp5p-GFP and Klp6p-GFP both localize to cytoplasmic microtubules throughout the cell cycle and to spindles in mitosis, but their localizations are not interdependent. During the meiotic phase of the life cycle, both of these kinesins are essential. Spore viability is low in homozygous crosses of either null mutant. Heterozygous crosses of klp5Delta with klp6Delta have an intermediate viability, suggesting cooperation between these proteins in meiosis.

The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds.

The cycle of spindle pole body (SPB) duplication, differentiation, and segregation in Schizosaccharomyces pombe is different from that in some other yeasts. Like the centrosome of vertebrate cells, the SPB of S. pombe spends most of interphase in the cytoplasm, immediately next to the nuclear envelope. Some gamma-tubulin is localized on the SPB, suggesting that it plays a role in the organization of interphase microtubules (MTs), and serial sections demonstrate that some interphase MTs end on or very near to the SPB. gamma-Tubulin is also found on osmiophilic material that lies near the inner surface of the nuclear envelope, immediately adjacent to the SPB, even though there are no MTs in the interphase nucleus. Apparently, the MT initiation activities of gamma-tubulin in S. pombe are regulated. The SPB duplicates in the cytoplasm during late G2 phase, and the two resulting structures are connected by a darkly staining bridge until the mitotic spindle forms. As the cell enters mitosis, the nuclear envelope invaginates beside the SPB, forming a pocket of cytoplasm that accumulates dark amorphous material. The nuclear envelope then opens to form a fenestra, and the duplicated SPB settles into it. Each part of the SPB initiates intranuclear MTs, and then the two structures separate to lie in distinct fenestrae as a bipolar spindle forms. Through metaphase, the SPBs remain in their fenestrae, bound to the polar ends of spindle MTs; at about this time, a small bundle of cytoplasmic MTs forms in association with each SPB. These MTs are situated with one end near to, but not on, the SPBs, and they project into the cytoplasm at an orientation that is oblique to the simple axis. As anaphase proceeds, the nuclear fenestrae close, and the SPBs are extruded back into the cytoplasm. These observations define new fields of enquiry about the control of SPB duplication and the dynamics of the nuclear envelope.

cut11(+): A gene required for cell cycle-dependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe.

The "cut" mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11(+) suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11(+) encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

pkl1(+)and klp2(+): Two kinesins of the Kar3 subfamily in fission yeast perform different functions in both mitosis and meiosis.

We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neither klp2(+) nor pkl1(+) is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles of cut7(ts), a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2 Delta,dhc1-d1 in karyogamy, pkl1 Delta,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1 Delta shortens it. The anaphase spindle of klp2 Delta becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.

Myelodysplasia, chemical exposure, and other environmental factors.

This paper describes a case-control study of the occupational and environmental exposures of patients with myelodysplasia. The methodology, first described in Canada for solid tumors, estimates lifetime exposures to a number of potential toxic hazards or carcinogens. This pilot study confirms that the methodology, with the use of questionnaires and interviews, can estimate exposures to specific chemicals and shows some significant associations with myelodysplasia, including exposure to petrol or diesel compounds.

Topoisomerase II alpha expression in acute myeloid leukaemia and its relationship to clinical outcome.

The treatment of acute myeloid leukaemia (AML) is often complicated by resistance to chemotherapeutic drugs. Many of the most effective drugs used to treat haematological malignancies target the nuclear enzyme topoisomerase II. Resistance to these drugs in vitro has been associated with quantitative and qualitative changes in the enzyme. In this study, we have investigated topoisomerase II mRNA expression in leukaemic blasts from 23 AML patients. Expression levels ranged from 1-47% relative to the haemopoietic cell line HL60 in 16 evaluable patients. Thirteen of 16 patients achieved complete remission (CR). We have therefore chosen ease of entry into CR as the most sensitive clinical correlate. Decreased topoisomerase II mRNA expression in vitro results in drug resistance. The clinical relevance of reduced expression is not known. All cases of AML and de novo AML have been studied separately. We are unable to identify a correlation between topoisomerase II mRNA levels and ease of entry into CR in either of these groups. Taking these findings into account, group size calculations have been undertaken and indicate that a multi-centre study of this question is warranted.

Identification of the lipophilic factor produced by macrophages that stimulates steroidogenesis.

Macrophages are known to release a lipophilic factor that stimulates testosterone production by Leydig cells. This macrophage-derived factor (MDF) is thought to be physiologically relevant, because removal of macrophages from the testis results in altered testosterone secretion and reduced fertility. The purpose of the present study was to purify this factor, elucidate its chemical structure, and determine whether it is both present in the testis and acts when injected intratesticularly. Culture media from testicular and peritoneal macrophages were extracted with ether, and the organic phase was sequentially purified on C18, silica, and cyano-HPLC columns. MDF was detected using a rat Leydig cell bioassay, with testosterone secretion being the end point. Purified material and crude ether extracts were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. The time of elution of MDF from both testicular and peritoneal macrophages was identical on all three HPLC columns. A single peak was observed when MDF, obtained from the final HPLC column, was analyzed by gas chromatography. The MS fragmentation pattern of purified material from both peritoneal and testicular macrophages was identical to that of a reference preparation of 25-hydroxycholesterol. Also, the nuclear magnetic resonance spectrum of MDF was similar to that of authentic 25-hydroxycholesterol. When 25-hydroxycholesterol was subjected to the identical purification scheme as MDF, it was found to elute at the same times as MDF on all three columns and elicited activity in the Leydig cell bioassay as expected. Control medium purified identically did not contain 25-hydroxycholesterol or have biological activity. Ether extracts of testis contained 25-hydroxycholesterol, indicating that this compound is present under physiological conditions. Similarly, when 25-hydroxycholesterol was injected into the testis of adult rats, testosterone production was increased within 3 h. Taken together, these data indicate that the lipophilic factor produced by macrophages that stimulates steroidogenesis is 25-hydroxycholesterol.

A case control study of factors associated with macroscopic breast cysts.

Women who have a breast cyst aspirated are at increased risk of developing breast cancer. We present an age-matched case control study comparing the reproductive characteristics of 352 women who had a breast cyst aspirated with a control group of 352 contemporaneous clinically normal women. Women with breast cysts were more likely to be nulliparous [odds ratio (OR) = 2.28, 95% confidence interval (CI) = 1.34-3.88] or have a late age at first live birth (chi 2 trend = 5.6, P less than 0.025), and a late menopause (chi 2 trend = 4.3, P less than 0.05). They were less likely to have ever used the oral contraceptive pill (OR = 0.38, 95% CI = 0.26-0.55) or to have used the pill for a short duration (chi 2 trend = 16.8, P less than 0.001), and were less likely to have had a hysterectomy (OR = 0.58, 95% CI = 0.36-0.93). They were more likely to wear a small bra (chi 2 trend = 18.6, P less than 0.001) and bra cup (chi 2 trend = 5.6, P less than 0.025). Nulliparity, late age at first live birth and late menopause are factors common to breast cancer risk.

Occupational exposures and haematological abnormalities among ordnance factory workers: a case control study.

The lifetime exposures to an extensive list of chemical and other potentially toxic hazards were estimated for all available employees at an ordnance factory by questionnaire-based interview. Exposure histories of 32 (of 33) previously diagnosed as haematologically abnormal (cases) were compared with 322 (of 345) normals (controls). Among 'ordnance factory chemicals', modestly increased odds ratios were observed for men for acetic anhydride (2.8), stearic acid (2.8), and possibly for resorcinol (2.9), TNT (2.4) and hydroxy terminated polybutadene (HTPB) (2.4). Increased odds ratios were also observed in exposures not directly related to ordnance manufacture, including mineral acids, welding fumes, exhaust gases and insecticides. While, small numbers in the case group limit the statistical significance of reported odds ratios, one should be cautious about committing a type II error. These findings may partly explain the previously reported unusual prevalence of haematological abnormalities within the factory.

Occupational and environmental exposures and myelodysplasia: a case-control study.

A case-control study of newly diagnosed myelodysplastic syndrome patients investigated lifetime exposures through occupation, environment or hobby by questionnaire, structured and semi-structured interview. The exposure histories of 400 individually matched pairs were compared. Increased or possibly increased odds ratios were observed for radiation (2.05, 95% confidence interval 1.16-3.76), halogenated organics (1.57, 0.97-2.57), metals (1.40, 0.99-2.00), several specific radiation exposures and individual chemicals and for childlessness (1.46, 1.01-2.11). Since myelodysplasia generally carries a poor prognosis, whether or not individuals convert to leukaemia or to other cancer, these findings add to previous reports of exposures implicated in the aetiology of leukaemia and add to the case for minimizing exposures to radiation and halogenated organics.

Effect of systemic vasodilators on internal mammary artery flow. Implications for postoperative treatment after myocardial revascularization.

Postoperative spasm of the internal mammary artery graft can cause morbidity and mortality after myocardial revascularization. To our knowledge, the ability of systemic vasodilators to overcome internal mammary artery spasm has not been studied clinically. In 50 patients in whom the left internal mammary artery was used for myocardial revascularization, we have investigated the effect of five agents on internal mammary artery free flow: normal saline, dobutamine, glyceryl trinitrate, sodium nitroprusside, and enoximone, a phosphodiesterase III inhibitor. After the internal mammary artery was harvested, free flow was measured under controlled hemodynamic conditions before any pharmacologic intervention (flow 1) and a mean of 18.5 +/- 3 (standard deviation) minutes after a systemic infusion of one of the five agents was begun (flow 2). The increase in free flow expressed as a percentage of initial flow was greater for enoximone (94% +/- 24%) than for normal saline (18% +/- 11%), dobutamine (40% +/- 27%), and glyceryl trinitrate (52% +/- 36%) (all three p < 0.01). The increase in flow for sodium nitroprusside (78% +/- 37%) was greater than for normal saline and dobutamine (both p < 0.05). We therefore recommend the systemic use of enoximone and sodium nitroprusside, in rank order, to prevent and treat postoperative spasm of the internal mammary artery.

Specificity of association in analysis of mortality and inference on causality.

Specificity of association between putative risk factor and disease under study is important to inference on causality. Nevertheless many studies investigate mortality of a single disease without comparison with a control. Age-standardized proportional mortality ratios make single disease studies into case-control studies and thus demonstrate whether or not associations are disease specific. Comparison of disease-specific with all-cause mortality experiences of whole populations classified by exposure, clearly distinguishes between exposures associated with more death and with earlier/younger death, thereby overcoming an important limitation of the familiar standardized mortality ratio (SMR). Smoking is associated with more death from lung cancer (lifetime cause-specific proportions, never 1%, light 6%, moderate 8% and heavy 12%) and with earlier/younger death from ischaemic heart disease (never 35%, light 34%, moderate 32% and heavy 29%).

Perinatal and infant mortality in Wales: inter-district variations and associations with socio-environmental characteristics.

Stillbirth rates, perinatal death rates, early and late neonatal death rates and (post-neonatal) infant death rates are reported for Wales since local government and NHS reorganization in 1974. The time trends in these rates show declining mortality, in full weight and in low birthweight babies. Analysis of average rates for the period 1974-81 inclusive in the 37 local authority districts within Wales demonstrate wide variations, with PMRs ranging from 11.5 to 22.5 per 1000. Many highly statistically significant associations were evident between socioeconomic characteristics of the districts and stillbirth rates but few with neonatal death rates and none with infant death rates.

An example of medical audit under political pressure.

Management of medical services necessitates decisions regarding the possibly ineffective treatment programmes, hospitals and (even) individual practitioners. Although rigorous evaluations of effectiveness can only be achieved by randomized trials there has been, in recent years, much increase in audit (or peer review) activity. Ideally medical audit is undertaken as a collaborative exercise, and follows preplanned methods of data collection and evaluation of outcome. However, real management situations frequently do not conform to the ideal, as when the medico-political finger of accusation points at one particular hospital and asks for an immediate enquiry. This audit analyses the mortality and morbidity after coronary artery bypass graft surgery in the 'suspect' hospital and in control hospitals. The paper describes the difficulties of obtaining comparable records post hoc and the interpretations of small differences, mostly non-significant statistically yet possibly 'real', both in the characteristics of patients operated on and in the outcomes of operations.

Mortality from ischaemic heart disease--inter-town variation and its association with climate in England and Wales.

In both seasonal and inter-town variation, the mortality from ischaemic heart disease in England and Wales in 1969-71 is very highly correlated with temperature. Also in the inter-town variation, IHD mortality is highly correlated with rainfall and with socio-economic index. It is postulated that the same mechanism of body cooling underlies seasonal, inter-town and socio-economic variations in IHD mortality. The first law of thermodynamics states the heat and energy are equivalent. Therefore to maintain body temperature against an increased thermal gradient (caused by a lower environmental temperature as in seasonal variation, or a temperature-rainfall interaction as in inter-town variation) more energy must be expended by the body or, alternatively, the body must reduce heat loss by increasing thermal insulation. It is to be expected that there will be many accompanying changes in physiological parameters and several of these are discussed.

Sudden infant death syndrome in Cardiff, association with epidemic influenza and with temperature - 1955-1974.

The effects of social class, season, low temperature and sudden falls in temperature are investigated in 286 cases of Sudden Infant Death Syndrome in Cardiff in the years 1955--1974. The association with low temperature was striking: unsuspected hypothermia may be an important feature of these deaths. Sudden infant death incidence was associated with influenza A but not influenza B epidemics.

Epidemiology for medical students: a course relevant to clinical practice.

A new course was designed to make epidemiology clinically relevant to medical undergraduates. The objectives were that students should (1) know the epidemiology of common diseases, (2) understand epidemiological concepts useful in diagnosis and treatment, and (3) be able to critically assess published medical evidence. Results of a written examination showed that objectives 1 and 2 had been 'easily' achieved by 80% and 68% of students respectively. Student opinion of the course, assessed by an anonymous questionnaire, showed that the majority of students considered the course to be an important part of medical education. Before the course 19% felt 'reasonably able' or 'very able' to achieve nine specific objectives related to epidemiological concepts in diagnosis and treatment. By the end of the course this had risen to 78%.

Epidemiology for medical students: a controlled trial of three teaching methods.

Medical students taking a course in epidemiology for clinical practice were taught by either lectures, small group seminars or self-learning packages. Examination performances were no different for the three groups, but self-perceived mastery of learning objectives, and satisfaction with the course were higher for students who received self-learning packages. Sixty per cent of self instruction students found the teaching method was successful compared with 37% of the seminar students and only 19% who received lectures. A combination of self-instructional package and seminar would seem to hold most promise for a workable and effective course.