Centrosome maturation.

In the past, centrosome maturation has been described as the change in microtubule nucleation potential that occurs as cells pass through specific phases of the cell cycle. It is suggested that the idea of centrosome maturation be expanded to include gain of functions that are not necessarily related to microtubule nucleation. Some of these functions could be transient and dependent on the temporary association of molecules with the centrosome as cells progress through the cell cycle. Thus, the centrosome may best be viewed as a site for mediating macromolecular interactions, perhaps as a central processing station within the cell. The centromatrix, a relatively stable lattice of polymers within the centrosome's PCM, could serve as a scaffold for the transient binding of mediator molecules, as well as allow the dynamic exchange of centrosome constituents with a soluble cytoplasmic pool. New evidence adds support to the idea that centrioles are crucial for the maintenance of PCM structure. However, significant evidence indicates that aspects of centrosome structure and function can be maintained in the absence of centrioles. In the case of paternal centrosome maturation, sperm centrioles may not contain an associated centromatrix. It is proposed that regulation of paternal centrioles or centriole associated proteins could mediate centriole-dependent centromatrix assembly following fertilization. Thus, regulation of centromatrix-centriole interactions could be involved in maintaining the integrity of the centrosome's PCM and play an important role in centrosome disassembly during cell differentiation and morphogenesis.

Isolation of centrosomes from Spisula solidissima oocytes.

We have described methods for the preparation of lysates and isolation of centrosomes from parthenogenetically activated oocytes of the surf clam, S. solidissima. Although oocyte availability is seasonal, between June and August as much as 2 liters of lysate can be generated by a single person. Since lysate can be stored frozen at -80 degrees C with no apparent loss in centrosome-dependent microtubule nucleation, this is a convenient system for year-round experimentation. On average, per milliliter of frozen-stored lysate, 2 or 3 x 10(6) centrosomes can be obtained at 3000- or 4000-fold purification by sucrose-density gradient centrifugation. Centrosome fractions typically contain 6.0 x 10(-12) g of protein per centrosome (Vogel et al., 1997) and 140-200 micrograms of protein is usually obtained from a single run involving six sucrose-density gradients (12 ml of lysate). One person can easily run three preparations in a day, and thus 420-600 micrograms of centrosome protein could be prepared daily. Therefore, based on the effort of one individual, as much as 20-40 mg of centrosome protein could be prepared per year. Another convenient feature of the system is that once centrosomes are isolated, they can be stored in high sucrose media at -80 degrees C for years with little or no loss in microtubule nucleation potential. Once isolated, centrosomes can be used for protein analysis, ultrastructural studies, or in functional reconstitution assays (Vogel, 1997). In addition, these preparations offer the isolation of sufficient quantities of centrosome proteins to be used as antigens for generating centrosome-specific antibodies or for obtaining protein sequence for the purpose of antibody production or the design of oligonucleotide primers for isolating cDNA fragments coding for centrosome proteins. Thus, the preparations described offer a biochemical approach for defining centrosome composition. The methods described for immunofluorescence analysis of asters assembled in lysates offer rapid and convenient preparations for screening antibodies for centrosome localization and specificity. Finally, the ability to prepare large quantities of homogeneous centrosomes should enhance ultrastructural studies since many centrosomes can be sectioned and analyzed simultaneously by EM, avoiding the problem of having to hunt through sections of single cells to find a single centrosome for analysis. In addition, colloidal gold localization studies, using antibodies and EM to pinpoint the relative location of individual proteins, could be carried out on populations of centrosomes in the same preparation simultaneously, thus drastically expanding the quantity of data gathered. In conclusion, the clam oocyte system described here offers the potential for a combined structural and biochemical approach for identification of novel centrosome proteins and elucidation of the molecular basis of microtubule nucleation, centrosome assembly, and centrosome function.

The effects of smoking on bone mass and the rates of bone loss among elderly Japanese-American men.

Bone density and bone loss rates were examined among Japanese-American men categorized as current cigarette smokers, past smokers, and nonsmokers. The design included a retrospective study of smoking and bone density and a prospective study of current smoking and bone loss rates. The mean length of follow-up was 5 years; the setting was the island of Oahu. The subjects included 1303 men in the Hawaii Osteoporosis Study, 51-82 years old at their initial examination. Twenty percent were current smokers, 45% past smokers, and 35% had never smoked. Their bone density was measured at the distal and proximal radius and calcaneus using single photon absorptiometry. Compared with never smokers, current and past smokers had significantly lower bone density, especially in the predominantly cancellous calcaneus (4.8 and 4.3% lower, respectively) and partially trabecular distal radius (1.8 and 3.3% lower, respectively). The magnitude of the smoking effect was linked strongly to the duration of smoking and also to the number of cigarettes smoked. Bone loss rates subsequent to the initial measurement were greater in the current smokers than the never smokers (20.5, 27.2, and 9.7% greater at the calcaneus, distal, and proximal radius, respectively) but the differences did not achieve significance. Smokers of more than one pack per day had 32.0, 77.6, and 30.7% greater loss rates than never smokers in these same sites; the difference achieved significance at the distal radius. The results from the distal radius suggest that these smokers may increase their fracture risk 10-30% per decade of smoking. The adverse effects of smoking appeared to be greater in cancellous than cortical bone.

Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin.

Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures approximately 25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

Factors in daily physical activity related to calcaneal mineral density in men.

To determine the factors in daily physical activity that influence the mineral density of the calcaneus, we recorded walking steps and the type and duration of exercise in 43 healthy 26-to 51-yr-old men. Areal (g.cm-2) calcaneal bone mineral density (CBMD) was measured by single energy x-ray densitometry (SXA, Osteon, Inc., Wahiawa, HI). Subjects walked a mean (+/- SD) of 7902 (+/- 2534) steps per day or approximately 3.9 (+/- 1.2) miles daily. Eight subjects reported no exercise activities. The remaining 35 subjects spent 143 (2-772) (median and range) min.wk-1 exercising. Twenty-eight men engaged in exercise activities that generate single leg peak vertical ground reaction forces (GRFz) of 2 or more body weights (high loaders, HL), and 15 reported exercise or daily activities that typically generate GRFz less than 1.5 body weights (low loaders, LL). CBMD was 12% higher in HL than LL (0.668 +/- 0.074 g.cm-2 vs 0.597 +/- 0.062 g.cm-2, P < 0.004). In the HL group, CBMD correlated to reported minutes of high load exercise (r = 0.41, P < 0.03). CBMD was not related to the number of daily walking steps (N = 43, r = 0.03, NS). The results of this study support the concept that the dominant factor in daily physical activity relating to bone mineral density is the participation in site specific high loading activities, i.e., for the calcaneus, high calcaneal loads.

When should bone density measurements be repeated?

We calculated how long to wait before repeating bone mineral density (BMD) measurements to reassess fracture risk. Correlation results from serial measurements of 495 postmenopausal Japanese-American women were used to estimate 95% confidence intervals (CI) for future BMD. After 7 years of follow-up, BMD correlations with the initial measurement ranged between 0.81 and 0.94, depending on age group and measurement site. In this analysis, the period between measurements was defined as the time required for the lower 95% CI to fall below the BMD value corresponding to doubling of fracture risk. Progressive bone loss causes fracture risk to double after 10 years, on average. However, the 95% CIs indicate that a second BMD measurement will detect risk doubling after only 2 or 3 years for some women. For untreated, early postmenopausal women, the period between measurements was approximately 2-5 years for the radius and 4-6 years for the calcaneus, depending on the initial BMD level. The period was approximately 1 year longer for women age 60 and older. Treatments that halve the bone loss rate would increase the period by 1-3 years. In the absence of a second measurement of BMD, the CI will continue to expand with time, corresponding to a wider range in risk between individuals, and a greater proportion of women will be at increased fracture risk. Obtaining a second BMD measurement pinpoints the patient's status within the precision of the measurement. We conclude that repeated BMD measurements will provide a more accurate estimate of fracture risk than a single, baseline measurement.

Expression of the two maize TATA binding protein genes and function of the encoded TBP proteins by complementation in yeast.

A single gene encodes the TATA binding protein (TBP) in yeasts and animals. Although two TBP-encoding genes (Tbp) previously were isolated from both Arabidopsis and maize, the expression and in vivo function of the encoded plant TBPs were not investigated. Here, we report that the two highly conserved maize Tbp genes are unlinked and reside within larger, ancestrally duplicated segments in the genome. We find quantitative differences in Tbp1 versus Tbp2 transcript accumulation in some maize tissues. These nonidentical expression patterns may indicate differences in the tissue-specific regulation of these genes, which might allow the two encoded maize TBP isoforms to perform nonoverlapping functions in the plant. In addition, we show that the maize TBP products, unlike animal TBPs, are functionally interchangeable with yeast TBP for conferring yeast cell viability. This is a conclusive demonstration of in vivo activity for a nonyeast TBP protein, and these complementation results point to particular amino acids in TBP that are likely to influence species-specific protein interactions.

Grossly abnormal liver-spleen scan in a patient with veno-occlusive disease of the liver that normalized completely on follow-up.

A case of veno-occlusive disease of the liver of unknown etiology is reported. In the case, the gross abnormality of liver parenchyma, ascites, and colloid shift to spleen, bones, and lungs found on a Tc-99m SC liver-spleen scan, and confirmed on liver biopsy, resolved completely on follow-up. Histologic correlation and a brief review of the literature are presented.

The determination of dual pancreatic and renal transplant graft vascular patency with Tc-99m HMPAO.

Combined pancreatic-renal transplants promise the restoration of physiologic control of serum glucose and normal renal function. As pancreatic transplantation becomes more common, there is an increased need for rapid, noninvasive evaluation of vascular graft patency and function. Pancreatic transplants share the renal transplant's complications of ischemia at harvest but are at greater risk. Tc-99m HMPAO is a lipophilic complex that clears rapidly from the blood after intravenous injection, and tissue accumulation is proportional to regional perfusion. Using Tc-99m HMPAO to monitor the vascular competency has the advantage of a high count rate during dynamic scintigrams but, in contrast to Tc-99m DTPA, has excellent delayed static images. Four patients who received combined cadaveric pancreatic-renal transplants and had a total of eight Tc-99m HMPAO scintigraphic examinations were reviewed.

Relationship between bone mass and rates of bone change at appendicular measurement sites.

The rate of bone change among postmenopausal women may vary depending upon the initial bone mass. Examining this possibility is difficult, however, because of a negative statistical bias that occurs when change is regressed against the initial value of the same variable. In this article, four statistical methods were applied to measure the association between bone mass and the rate of bone change. The study population was Japanese-American women, who were monitored for approximately 5 years. Bone changes were determined for the calcaneus and the distal and proximal radius. The results were consistent across the bone sites but differed between statistical methods. Three of the four methods indicated that the women with the greater bone mass had the greater loss rates. The fourth method did not support this association. Possible reasons for the discordant results are discussed. Using the "best" estimate of the relationship, a gradual convergence of bone mass was projected over time toward the population mean. The convergence occurred because women with higher bone mass had a somewhat faster loss rate than women with lower bone mass. Overall, however, the variation in bone mass between individuals was large compared to the rate of convergence.

Comparison of left anterior oblique, anterior and geometric mean methods for determining gastric emptying times.

The accurate determination of gastric emptying time requires correction or compensation for tissue attenuation. The gold standard for tissue attenuation correction for gastric emptying is the geometric mean of the gastric counts from the anterior and posterior views. For reasons of efficiency, many community hospitals acquire only the anterior projection. This study addressed the hypothesis that, using the left anterior oblique view alone, one can minimize the effect of variation in attenuation as the meal moves from the fundus to the stomach to the more anterior antrum to a degree equal to that of the geometric mean technique. We studied 42 consecutive patients using a standardized 300-g meal labeled with 650 muCi of 99mTc-sulfur colloid. The patients were imaged in the anterior (ANT), posterior (POST) and left anterior oblique (LAO) views every 15 min for 90 min. Linear regressions were obtained using the ANT, LAO and GM data. Cross-correlation of the T1/2 for 35 cases showed an R value for the GM versus LAO of 0.95 and GM versus ANT of 0.84. The p value greater than 0.49, for the paired two-tailed t-test of the LAO and GM methods. The p value for the ANT and GM methods is 0.0058 indicating a significant difference between these methods. The cross-correlation, F-test p and t-test p values support the hypothesis that there is no significant difference between the geometric mean and left anterior oblique gastric emptying times. It is therefore reasonable to substitute the left anterior oblique for routine GET when using a solid meal in patients with normal gastric anatomy, albeit altered physiology.

The TATA box promoter region of maize Adh1 affects its organ-specific expression.

We have isolated two lineage-related Mutator (Mu3) transposon-induced Adh1 promoter mutants in maize: Adh1-3F1124 carries a duplicated TATA box and its revertant, Adh1-3F1124r17, bears a deleted TATA box. Both alterations lead to unique patterns of organ-specific ADH1 enzyme expression. Enzyme activity in Adh1-3F1124 sporophytic organs (scutellum and roots) is greatly reduced, while activity levels remain normal in the male gametophyte (pollen). Conversely, enzyme activity in Adh1-3F1124r17 roots and scutellum is partially restored, but is concomitantly reduced in pollen. Transcript analysis suggests (i) that the TATA box region of the Adh1 gene influences post-transcriptional processes in the male gametophyte but not in roots and (ii) that organ-specific transcription signals in the promoter are distinct from the previously identified anaerobic environment-specific cis-acting transcription signals. Different organs appear to provide surrogate TATA function in different ways, leading to organ-specific differences in the length of the Adh1 message 5' leader.

Age-related changes in bone mass among Japanese-American men.

Bone mass and appendicular bone loss rates were examined in a cohort of Japanese-American men. Across their age range (ages 61 to 82 years) bone mass steadily declined at the proximal and distal radius, and at the calcaneus. The cross-sectional reduction in bone mass was 3.5-6.3% per decade at the various sites. Longitudinal measurements of the same cohort indicated greater losses than suggested by the cross-sectional data, yet still less than 10% per decade. Linear trends of increasing loss rates with aging were significant at the calcaneus, and marginally significant at the radius sites. However, the oldest men in the cohort strongly influenced these trends. Men under age 75 had essentially constant annual rates of bone loss. The most elderly men had both the lowest bone mass and the greatest bone loss rates.

Long-term precision of bone loss rate measurements among postmenopausal women.

Repeated measurements of bone mineral content can indicate the rate of bone loss among postmenopausal women. The clinical utility of such loss rate measurements will depend upon the long-term precision of the measurements. We have analyzed the precision of appendicular bone measurements among 495 Japanese-Americans followed for an average of 5.3 years and of both appendicular and axial measurements among 70 clinical trial participants followed for 2 years. Tables were derived from these analyses to quantitate the precision of individual loss rates under varying measurement conditions that might be encountered in clinical practice. The results demonstrate that only unusually rapid loss rates could be identified with confidence within short intervals, such as 1 year or 2. Extending the length of follow-up, however, appreciably improved the measured loss rate precision. In comparisons between bone sites, appendicular sites were determined to achieve a specified precision within the shortest intervals, followed by spine dual photon absorptiometry measurements. Spine quantitative computerized tomography measurements and measurements of hip sites required considerably longer follow-up intervals to achieve comparable precision.

The clinical application of serial bone mass measurements.

Although the short-term precision of various bone mineral content (BMC) measurements is known, questions about the clinical use of serial BMC measurements remain: how frequently should BMC be measured? When is it appropriate to calculate bone loss rates? How are estimates of loss rate interpreted? This paper discusses both biological and technical sources of uncertainty, and the estimation of confidence limits for measured bone loss rates. For many, possibly most, patients, calculation of bone loss rate may not be necessary; however, repeated measures of BMC can still be useful for re-evaluating fracture risk. Indications for repeating BMC measurements may include low initial BMC (moderate to high fracture risk), anticipation of rapid bone loss (e.g., menopause, estrogen discontinuation), and verification of treatment efficacy.

Changes in body fluid compartments during a 28-day bed rest.

The extravascular fluid responses to real or simulated space-flight are not well-documented. In this study serial isotope measurements were used to obtain measurements of the body fluid responses of 10 22-29-year-old men during 28 d of simulated microgravity (bed rest). The subjects were maintained on a controlled metabolic diet for 7 d before the study, during 14 d of ambulatory control, 28 d of horizontal bed rest, and 14 d of ambulant recovery. Fluid compartments were measured on control days 1 and 9, bed rest days 2, 14, and 28, and recovery days 7 and 14. By day 2 of bed rest, plasma volume (PV) and extra-cellular volume (ECV) decreased significantly by an average 209 and 533 ml, respectively. Red cell volume (RCV) and total body water (TBW) decreased more slowly, with average losses of 128 and 1,316 ml, respectively, after 28 d of bed rest. Early in the bed rest, TBW loss was mostly from the ECV. Thereafter, the TBW deficit was derived from the intracellular compartment, which decreased an average of 838 ml after 28 d. These results suggest losses from all fluid compartments during bed rest, with no evidence of restoration of ECV after 1-2 weeks.

Vertebral dimension differences between Caucasian populations, and between Caucasians and Japanese.

Various criteria have been proposed for using vertebral measurements to identify vertebral fractures. It is known that the normal distributions of vertebral heights and ratios vary with location within the spine. However, very little is known regarding the degree to which differences in these parameters may exist between populations. We report the vertebra-specific distributions of vertebral dimensions and ratios for Japanese-Americans, and compare these values to published data for Caucasians. The mean Japanese vertebral heights were 1 to 2 mm shorter than Caucasians, which may be due in part to the shorter stature of Japanese. However, differences in mean values were also observed between Caucasian populations. Furthermore, anterior/posterior vertebral height ratios differed between Caucasian studies, and between races. Additional studies are needed to determine to what degree these differences are due to technical and biological factors before criteria derived from one population can be used for identifying vertebral fractures in other populations of the same, or different, race.

Characteristics of respondents and nonrespondents in a prospective study of osteoporosis.

During 1981-1982, a cohort of elderly Japanese Americans living in Hawaii was recruited for an epidemiologic study of osteoporosis. The male subjects were simultaneously being examined for an epidemiologic study of heart disease. Baseline data collected from both the men and women at a previous heart disease examination were used to compare responders vs nonresponders. The target population for the osteoporosis study consisted of 1685 men and 1594 women. Of these, 1379 men (81.8%) and 1105 women (72.0%) participated in the initial osteoporosis examination. For each sex, nonrespondents were older and had higher systolic blood pressure levels than did the respondents. Male nonresponders had a higher stroke prevalence and more frequent recent use of vasodilator medicine. Female nonresponders had a less frequent history of having ever taken female hormones than did the responders. The responders and nonresponders were reasonably similar in other respects, as indicated by the comparison of more than 40 other variables. This suggests that nonresponse bias is probably not a major influence in exposure-disease associations in this osteoporosis cohort. We believe this is the first published report dealing with nonresponse characteristics in a cohort study of osteoporosis.