OBJECTIVE

To examine the role of tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta in collagen-induced arthritis (CIA), immediately after onset and during the phase of established arthritis.

METHODS

Male DBA/1 mice with collagen-induced arthritis were treated with antibodies against murine TNF alpha and IL-1 alpha/beta at different time points of the disease. IL-1 receptor antagonist (IL-1Ra) was administered using Alzet osmotic minipumps. The effect of anticytokine treatment was monitored by visual scoring. Histology and cytokine reverse transcription polymerase chain reaction (RT-PCR) analyses were performed at the end of the treatment period.

RESULTS

Anti-TNF alpha treatment showed efficacy shortly after onset of the disease, but had little effect on fully established CIA. Histologic analysis after early treatment revealed that anti-TNF alpha significantly reduced joint pathology, as determined by infiltration of inflammatory cells and cartilage damage. Anti-IL-1 alpha/beta treatment ameliorated both early and full-blown CIA. This clear suppression of established arthritis was confirmed by administration of high doses of IL-1Ra. Dose-response experiments showed that a continuous supply of 1 mg/day was needed for optimal suppression. Histologic analysis showed markedly reduced cartilage destruction both in the knee and the ankle joints. Autoradiography demonstrated full recovery of chondrocyte synthetic function of articular cartilage. In addition, we found that the IL-1 beta isoform plays a dominant role in established CIA. Profound suppression of CIA was observed with anti-IL-1 beta, although elimination of both IL-1 alpha and IL-1 beta still gave better protection. Analysis of messenger RNA with RT-PCR revealed that IL-1 beta was highly upregulated in synovium and cartilage at late stages of CIA, whereas anti-IL-1 beta treatment markedly reduced IL-1 beta message in the synovium.

CONCLUSIONS

The present study identified different TNF alpha/IL-1 dependencies in various stages of CIA and revealed that blocking of TNF alpha does not necessarily eliminate IL-1. Continuous, high doses of IL-1Ra are needed to block CIA.

Two infectious drug-resistance (R) factors, R28K and R6K, each conferring resistance to a number of penicillins by the synthesis of a penicillinase, were transferred to Proteus mirabilis PM1 and Escherichia coli RC85 host strains. Deoxyribonucleic acid (DNA) extracted from these strains was separated by density-gradient centrifugation and subjected to electron microscopy by use of a modification of the protein-monolayer diffusion technique. Analytical density-gradient centrifugation of the purified DNA from PM1 strains showed, in addition to the major peak at a density of 1.698 g/cm(3) characteristic of Proteus chromosomal DNA, a single satellite band at a density of 1.710 g/cm(3) [guanine plus cytosine (GC) base ratio 50%] for R28K and at 1.704 g/cm(3) (GC base ratio 45%) for R6K. Direct CsCl density-gradient centrifugation of crude lysates of the E. coli (R28K)(+) strain in the presence of ethidium bromide gave rise to a sedimentation profile with a single satellite peak containing covalently closed circular (CCC) DNA molecules with a mean contour length of 21.4 mum [44 x 10(6) atomic mass units (AMU)], although a minority was 13.6 mum in length. From the size of the major class, it was estimated that there were two to three copies of the R28K factor present as CCC molecules per chromosome at various phases of cellular growth. Similar studies of the E. coli (R6K)(+) lysates showed two satellite peaks; peak I contained mostly CCC molecules of contour length 12.8 mum (26 x 10(6) AMU), and peak II, intermediate to peak I and the chromosomal peak, contained CCC molecules of a similar size, together with about equal numbers of catenated molecules, mostly dimers consisting of two interlocked monomers of 12.8 mum. A smaller number (ca. 0.1%) of higher catenanes was also seen. The number of CCC copies of the R6K factor per chromosome present in peak I was calculated as 13 at logarithmic phase and 38 at stationary phase. In peak II, a constant ratio of about one catenated dimer per chromosome was found at all phases of growth. Penicillinase assays of cultures at different phases of growth showed a correlation between the estimated number of R-factor copies present as CCC molecules and the enzyme activity per cell for both R28K and R6K.

Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded 'introns first' held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes.

OBJECTIVE

To examine the role of peak bone mass and subsequent postmenopausal bone loss in the development of osteoporosis and the reliability of identifying women at risk from one bone mass measurement and one biochemical assessment of the future bone loss.

METHODS

Population based study.

METHODS

Outpatient clinic for research into osteoporosis.

METHODS

178 healthy early postmenopausal women who had participated in a two year study in 1977. 154 of the women underwent follow up examination in 1989, of whom 33 were excluded because of diseases or taking drugs known to affect calcium metabolism.

METHODS

Bone mineral content of the forearm and values of biochemical markers of bone turnover.

RESULTS

The average reduction in bone mineral content during 1977-89 was 20%, but the fast losers had lost 10.0% more than had the slow loser group (mean loss 26.6% in fast losers and 16.6% in slow losers; p less than 0.001). Prediction of future bone mineral content using baseline bone mineral content and estimated rate of loss gave results almost identical with the actual bone mineral content measured in 1989. Seven women had had a Colles' fracture and 20 a spinal compression fracture. The group with Colles' fracture had low baseline bone mineral content (34.7 (95% confidence interval 31.3 to 38.1) units v 39.4 (38.1 to 40.8) units in women with no fracture) whereas the group with spinal fracture had a normal baseline bone mineral content (38.1 (35.0 to 41.1) units) but an increased rate of loss (-2.4 (-3.5 to -1.3)%/year v -1.8 (-2.1 to -1.5)%/year in women with no fracture).

CONCLUSIONS

One baseline measurement of bone mass combined with a single estimation of the rate of bone loss can reliably identify the women at menopause who are at highest risk of developing osteoporosis later in life. The rate of loss may have an independent role in likelihood of vertebral fracture.

From a database containing the published nuclear hormone receptor (NR) sequences I constructed an alignment of the C, D and E domains of these molecules. Using this alignment, I have performed tree reconstruction using both distance matrix and parsimony analysis. The robustness of each branch was estimated using bootstrap resampling methods. The trees constructed by these two methods gave congruent topologies. From these analyses I defined six NR subfamilies: (i) a large one clustering thyroid hormone receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator-activated receptors (PPARs), vitamin D receptors (VDRs) and ecdysone receptors (EcRs) as well as numerous orphan receptors such as RORs or Rev-erbs; (ii) one containing retinoid X receptors (RXRs) together with COUP, HNF4, tailless, TR2 and TR4 orphan receptors; (iii) one containing steroid receptors; (iv) one containing the NGFIB orphan receptors; (v) one containing FTZ-F1 orphan receptors; and finally (vi) one containing to date only one gene, the GCNF1 orphan receptor. The relationships between the six subfamilies are not known except for subfamilies I and IV which appear to be related. Interestingly, most of the liganded receptors appear to be derived when compared with orphan receptors. This suggests that the ligand-binding ability of NRs has been gained by orphan receptors during the course of evolution to give rise to the presently known receptors. The distribution into six subfamilies correlates with the known abilities of the various NRs to bind to DNA as homo- or heterodimers. For example, receptors heterodimerizing efficiently with RXR belong to the first or the fourth subfamilies. I suggest that the ability to heterodimerize evolved once, just before the separation of subfamilies I and IV and that the first NR was able to bind to DNA as a homodimer. From the study of NR sequences existing in vertebrates, arthropods and nematodes, I define two major steps of NR diversification: one that took place very early, probably during the multicellularization event leading to all the metazoan phyla, and a second occurring later on, corresponding to the advent of vertebrates. Finally, I show that in vertebrate species the various groups of NRs accumulated mutations at very different rates.

The purpose of this study was to establish a nude rat orthotopic (organ-specific) human colorectal cancer model as an in vivo secondary screen for general evaluation of new anticancer agents against colorectal cancer and to evaluate practically the antitumor activity of 1 M tegafur-0.4 M 5-chloro-2,4-dihydroxypyridine-1 M potassium oxonate (S-1), a new p.o. fluoropyrimidine, in comparison to 1 M tegafur-4 M uracil [(UFT) effective on colorectal tumor in clinical]. After implantation of KM12C, a human colorectal cancer cell line, into the subserosal layer of the colon as a single-cell suspension, extensive local tumor growth and invasion to both the mucosal and the serosal sides were observed in all rats. Metastatic foci were also formed in both lymph nodes and lungs following local tumor growth in all of them. Using this method, an equitoxic dose of S-1 (15 mg/kg/day) and UFT (30 mg/kg/day) was administered p.o. for 14 consecutive days from 7 days after tumor cell implantation. S-1 showed a higher tumor growth inhibition than UFT did [S-1, 57% (significantly different from the tumor weight of the untreated group at P < 0.05) and UFT, 18% (P>> 0.05)]. When both drugs were administered to nude rats bearing KM12C injected into the cecal wall for 28 consecutive days at equitoxic doses, the mean survival in the S-1 group was 16 days longer than that in the untreated group (P < 0.01) but that in the UFT group was only 8 days longer (P>> 0.05). After the administration of an equitoxic dose of both drugs, S-1 gave the higher levels than UFT in various pharmacokinetic parameters as follows: area under the curve 0-24 h of 5-fluorouracil in plasma (3.5-fold), area under the curve 0-24 h of 5-fluorouracil incorporated into RNA in the tumor (1.3-fold), and thymidylate synthase inhibition rate (percentage) in the tumor (about 20%). Collectively, these findings suggested that this orthotopic human colorectal tumor model in nude rats is useful to evaluate the clinical therapeutic efficacy of drugs or therapies for colorectal cancer, and that S-1 had a higher therapeutic effect on human colorectal tumor than UFT did.

BACKGROUND

There is increasing concern about the quality, reliability, and independence of practice guidelines. Because no information is available on the methodological quality of the guidelines developed by specialty societies, we undertook a survey on those published in peer-reviewed journals.

METHODS

Practice guidelines produced by specialty societies and published in English between January, 1988, and July, 1998, where identified through MEDLINE. Their quality was assessed in terms of whether they reported: the type of professionals and stakeholders involved in the development process; the strategy to identify primary evidence; and an explicit grading of recommendations according to the quality of supporting evidence.

RESULTS

Overall, 431 guidelines were eligible for the study. Most did not meet the criteria: 67% did not report any description of the type of stakeholders, 88% gave no information on searches for published studies, and 82% did not give any explicit grading of the strength of recommendations. There was improvement over time for searches (from 2% to 18%, p<0.001) and explicit grading of evidence (from 6% to 27%, p<0.001). All three criteria for quality were met in only 22 (5%) guidelines.

CONCLUSIONS

Despite improvement over time, the quality of practice guidelines developed by specialty societies is unsatisfactory. Explicit methodological criteria for the production of guidelines shared among public agencies, scientific societies, and patients' associations need to be set up. Common standards of reporting, following the same principles that led to the CONSORT statement for randomised clinical trials, should be promoted.

Isolated cells from rat lacrimal glands were studied with the tight-seal whole-cell recording technique. Cells were dialysed with K-free solutions containing a high concentration of Ca2+ buffer in order to record Ca-dependent Cl- currents at a Ca2+ level fixed between 0.1 and 10 microM. HEDTA was preferable to EGTA as a Ca2+ buffer, even under conditions of equivalent equilibrium buffering power. After replacement of all internal K+ with Na+, the cells displayed a small conductance component which could be abolished by removal of external K+ or by external application of 2 mM-tetraethylammonium. It is suggested that this conductance is due to Ca-dependent K+ channels. The main part of the cell current was Cl selective. The Cl- conductance was negligible at 0.5 microM-Ca2+, and fully activated at 2 microM-Ca2+. The dose-response curve relating Cl- currents to the internal Ca2+ concentration, [Ca]i, was steeper than predicted by a simple binding isotherm reaction. Relaxations observed in response to voltage jumps could, in most cases, be fitted with single exponentials. At [Ca]i 0.5 microM, the curve relating the relaxation time constant, tau, to the membrane potential, displayed a maximum near +20 mV and 250 ms. At hyperpolarized potentials, tau varied by an e-fold factor in 130 mV. At [Ca]i 1 microM, tau decreased from 100 ms at -120 mV to 60 ms at +60 mV. Relaxation analysis gave an estimate of the variation of the channel open state probability, Po, with potential. At [Ca]i 0.5 microM, Po varied by an e-fold factor in 50-70 mV at hyperpolarized potentials, and saturated above +60 mV. At [Ca]i 1 microM, Po varied e-fold in 100 to 110 mV at hyperpolarized potentials, and saturated near +20 mV. External Cl- was substituted with various anions. From reversal potential measurements, the following permeability sequence was obtained: I- greater than NO3- greater than Br- greater than Cl- greater than F- greater than isethionate, methanesulphonate greater than glutamate. The corresponding normalized permeability coefficients were 2.7, 2.4, 1.6, 1, 0.2, 0.1, 0.1, 0.05. Replacement of external Cl- with Br-, isethionate, methanesulphonate or glutamate did not alter current kinetics as obtained during or after a depolarizing voltage jump.(ABSTRACT TRUNCATED AT 400 WORDS)

Array-based comparative genomic hybridization (CGH) measures copy-number variations at multiple loci simultaneously, providing an important tool for studying cancer and developmental disorders and for developing diagnostic and therapeutic targets. Arrays for CGH based on PCR products representing assemblies of BAC or cDNA clones typically require maintenance, propagation, replication, and verification of large clone sets. Furthermore, it is difficult to control the specificity of the hybridization to the complex sequences that are present in each feature of such arrays. To develop a more robust and flexible platform, we created probe-design methods and assay protocols that make oligonucleotide microarrays synthesized in situ by inkjet technology compatible with array-based comparative genomic hybridization applications employing samples of total genomic DNA. Hybridization of a series of cell lines with variable numbers of X chromosomes to arrays designed for CGH measurements gave median ratios for X-chromosome probes within 6% of the theoretical values (0.5 for XY/XX, 1.0 for XX/XX, 1.4 for XXX/XX, 2.1 for XXXX/XX, and 2.6 for XXXXX/XX). Furthermore, these arrays detected and mapped regions of single-copy losses, homozygous deletions, and amplicons of various sizes in different model systems, including diploid cells with a chromosomal breakpoint that has been mapped and sequenced to a precise nucleotide and tumor cell lines with highly variable regions of gains and losses. Our results demonstrate that oligonucleotide arrays designed for CGH provide a robust and precise platform for detecting chromosomal alterations throughout a genome with high sensitivity even when using full-complexity genomic samples.

OBJECTIVE

To determine neural correlates of recovery from aphasia after left frontal injury.

METHODS

The authors studied the verbal performance of patients with infarcts centered in the left inferior frontal gyrus (IFG), using a battery of attention-demanding lexical tasks that normally activate the left IFG and a simpler reading task that does not normally recruit the left IFG. The authors used positron emission tomography (PET) and functional MRI (fMRI) to record neural activity in the same group of patients during word-stem completion, one of the attention-demanding lexical tasks. To identify potential neural correlates of compensation/recovery, they analyzed the resulting data for the group as a whole (PET, fMRI) and also for each participant (fMRI).

RESULTS

Patients with damage to the left IFG were impaired on all attention-demanding lexical tasks, but they completed the word-reading tasks normally. The imaging studies demonstrated a stronger-than-normal response in the right IFG, a region homologous to the damaged left IFG. The level of activation in the right IFG did not correlate with verbal performance, however. In addition, a perilesional response within the damaged left IFG was localized in the two patients who gave the best performance in the word-stem completion task and showed the most complete recovery from aphasia.

CONCLUSIONS

Right-IFG activity may represent either the recruitment of a preexisting neural pathway through alternative behavioral strategies or an anomalous response caused by removal of the left IFG. Perilesional activity in the left IFG may represent sparing or restoration of normal function in peri-infarctual tissue that was inactive early on after injury. This activity may be of greater functional significance than right IFG activity because it was associated with more normal verbal performance.

This paper is a systematic review of research data on associations between physical activity and weight gain, with or without prior weight reduction. The selected studies were restricted to Caucasian (white) adults. Most studies with data on physical activity collected at follow-up, found an inverse association between physical activity and long-term weight gain. This finding was present in studies both with and without prior weight reduction. Prospective studies with physical activity measured at baseline, and randomized weight reduction interventions, gave inconsistent results regarding the effects of increased physical activity on weight change. The weighted mean weight regain in randomized studies with or without exercise training was 0.28 and 0.33 kg/month, respectively. Based on observational studies, it seemed that an actual increase in energy expenditure of physical activity of approximately 6300-8400 kJ/week (1500-2000 kcal/week) is associated with improved weight maintenance. This is more than was prescribed in most randomized trials, and certainly more than the participants actually achieved. Adherence to a prescribed exercise programme remains a big challenge. Before new methods to improve exercise adherence are found, the role of prescribed physical activity in prevention of weight gain remains modest.

Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI(q)-lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZalpha. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens, a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.

METHODS

Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, fever, central nervous system abnormalities, and renal dysfunction. In early reports the mortality approached 100 percent. A treatment protocol was introduced in 1979 for patients admitted to Johns Hopkins Hospital with the diagnosis of TTP-HUS. Treatment regimens included 200 mg of prednisone a day, for patients with minimal symptoms and no central nervous system symptoms, and prednisone plus plasma exchange, for patients with rapid clinical deterioration who did not improve after 48 hours of prednisone alone and for patients presenting with central nervous system symptoms and rapidly declining hematocrit values and platelet counts.

RESULTS

A total of 108 patients were treated, and 91 percent survived. Prednisone alone was judged to be effective in 30 patients with mild TTP-HUS (two relapses and two deaths). Plasma exchange plus prednisone was given to 78 patients with complicated TTP-HUS, resulting in 67 relapses and 8 deaths. Relapses occurred in 22 of 36 patients given maintenance plasma infusions. Neither splenectomy nor treatment with aspirin and dipyridamole was effective in those with a poor response to plasma exchange. None of the 71 patients tested had positive cultures for O157:H7 Escherichia coli. Nine percent of the patients were pregnant, and none gave birth to infants with TTP-HUS.

CONCLUSIONS

Effective treatment with 91 percent survival is available for patients with TTP-HUS.

1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.

Antibiotics with new mechanisms of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics based on the antimicrobial peptide protegrin I. Several rounds of optimization gave a lead compound that was active in the nanomolar range against Gram-negative Pseudomonas spp., but was largely inactive against other Gram-negative and Gram-positive bacteria. Biochemical and genetic studies showed that the peptidomimetics had a non-membrane-lytic mechanism of action and identified a homolog of the beta-barrel protein LptD (Imp/OstA), which functions in outer-membrane biogenesis, as a cellular target. The peptidomimetic showed potent antimicrobial activity in a mouse septicemia infection model. Drug-resistant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications.

Accurate estimation of the number of ovarian follicles at various stages of development is an important indicator of the process of folliculogenesis in relation to the endocrine signals and paracrine/autocrine mechanisms that control the growth and maturation of the oocytes and their supporting follicular cells. There are 10-fold or greater differences in follicular numbers per ovary at similar ages and/or strains reported in earlier studies using various methods, leading to difficulties with interpretation of ovarian function in control vs experimental conditions. This study describes unbiased, assumption-free stereological methods for quantification of early and growing follicular numbers in the mouse ovary. A fractionator approach was used to sample a defined fraction of histological sections of adult wild-type ovaries. Primordial and primary follicles were counted independently with the optical and physical disector methods. The fractionator/disector methods, which are independent of follicular size or shape, gave estimations of 1930 +/- 286 (S.E.M.) and 2227 +/- 101 primordial follicles, and 137 +/- 25 and 265 +/- 32 primary follicles per ovary at 70 and 100 days of age respectively. From exact counts on serial sections, secondary and later follicular numbers at 100 days of age were estimated at 135 per ovary. Remnants of zona pellucidae (a marker of previous follicular atresia) were estimated using a fractionator/physical disector approach and were approximately 500 per ovary. The application of the quantitative methods described will facilitate an improved understanding of follicular dynamics and the factors that mediate their growth and maturation and allow for a better comparison between different studies.

Data from 576 patients with papillary thyroid cancer were retrospectively analyzed. With a median follow-up of 10 years and three months, there were six deaths from, and 84 recurrences of, thyroid cancer. Of the latter, 16 (19 percent) could not be eradicated. Death from thyroid cancer occurred only in those 30 years of age or over at the time of diagnosis and only in patients with primary tumors larger than 1.5 cm in diameter. Locally invasive tumor was associated with a poor prognosis. Cervical lymph node metastases found at initial surgery were associated with higher recurrence rates but not higher mortality rates. Treatment with total thyroidectomy, postoperative radioiodine and thyroid hormone resulted in the lowest recurrence and mortality rates except in those patients with small primary tumors (less than 1.5 cm diameter) in whom less than total thyroidectomy and postoperative therapy with thyroid hormone alone gave results which did not differ statistically from those achieved with more aggressive therapy. No important differences in outcome were observed when cervical lymph node metastases were simply excised or more aggressively treated by neck dissection. External radiation and as initial adjunctive therapy adversely influenced outcome.

The steady-state kinetic properties of single Cl- channels with fast kinetics active at resting membrane potentials in cultured rat skeletal muscle were studied using the patch-clamp technique. Membrane patches containing single active Cl- channels were often observed, and binomial analysis of the percentage open time in membrane patches containing several Cl- channels indicated that the channels did not occur as obligatory dimers and that they gated independently of one another. Channel activity could be divided into three categories: normal, which included about 99% of the openings and closings; buzz mode, which included about 1% and consisted of bursts of about 50 brief open and shut intervals; and inactivated shut states which included about 0.01% of the shut intervals and lasted for seconds, and occasionally minutes. The method of maximum likelihood was used to determine the number of significant exponential components required to fit the distributions of open and shut intervals during normal activity. Open interval distributions required at least two components, with time constants of 0.52 and 1.5 ms at -40 mV and 7.6 degrees C. Shut interval distributions required at least five exponential components, with time constants of 0.064, 0.72, 1.9, 12.3 and 350 ms. Kinetic reaction schemes were developed for the normal and buzz mode using maximum likelihood techniques to determine the most likely models and rate constants. In developing these models the effects of limited time resolution and missed events were taken into account. Each model tested typically had two or more sets of equally likely rate constants. Incorrect sets of rate constants resulting from the effect of missed events could be eliminated by analysis of the data with different time resolutions. Normal activity could be accounted for by several different seven-state models with two open and five shut states. As different models could be found that gave identical descriptions of the data, the distributions of open and shut intervals were not sufficient to define a unique model. It was established that no other seven-state models would be found that describe the distributions of open and shut intervals during normal activity better than the most likely presented models.(ABSTRACT TRUNCATED AT 400 WORDS)

The performance of 14 different recombination detection methods was evaluated by analyzing several empirical data sets where the presence of recombination has been suggested or where recombination is assumed to be absent. In general, recombination methods seem to be more powerful with increasing levels of divergence, but different methods showed distinct performance. Substitution methods using summary statistics gave more accurate inferences than most phylogenetic methods. However, definitive conclusions about the presence of recombination should not be derived on the basis of a single method. Performance patterns observed from the analysis of real data sets coincided very well with previous computer simulation results. Previous recombination inferences from some of the data sets analyzed here should be reconsidered. In particular, recombination in HIV-1 seems to be much more widespread than previously thought. This finding might have serious implications on vaccine development and on the reliability of previous inferences of HIV-1 evolutionary history and dynamics.

Molecular marker loci were used to investigate the inheritance of morphological traits that distinguish maize (Zea mays ssp. mays) from a closely related wild relative, teosinte (Z. mays ssp. mexicana). Regression and interval mapping analyses gave largely congruent results concerning the numbers of loci controlling the morphological traits and the magnitudes of their effects; however, interval mapping tended to give larger estimates for the magnitudes of the effects of the morphological trait loci. This tendency was exaggerated for traits that were non-normally distributed. Variation for most inflorescence traits is controlled by one or two regions of the genome with large effects plus several other regions with relatively small effects. As such, the data are congruent with a mode of inheritance for most traits involving one or two major loci plus several minor loci. Regions of the genome with large effects on one trait consistently had smaller effects on several other traits, possibly as a result of pleiotropy. Most of the variation for the dramatic differences in inflorescence morphology between maize and teosinte is explained by five restricted regions of the genome. One of these regions encompasses a previously described gene, tb1 (teosinte branched), and the effects of this region on inflorescence architecture are similar to the known effects of tb1. Implications of this work for the genetic basis of morphological evolution in plants are discussed.

373 (59%) out of 636 faecal specimens obtained during the first 2 years of life of 72 Mexican children yielded adherent Escherichia coli (HEp-2 cells). Strains with localised adherence were significantly associated with acute non-bloody diarrhoea, whereas strains with aggregative adherence were significantly associated with persistent diarrhoea. Half the strains with localised adherence were not enteropathogenic E coli serotypes nor did they hybridise with an enteropathogenic E coli adherence factor DNA probe. All strains with localised adherence gave a positive fluorescent actin staining (FAS) assay, irrespective of serotype. One-third of children colonised by aggregative strains had bloody diarrhoea. Isolation of strains with diffuse adherence was not related to type or duration of diarrhoea but was generally associated with isolation of another pathogenic organism.

The rice genus, Oryza, which comprises 23 species and 9 recognized genome types, represents an enormous gene pool for genetic improvement of rice cultivars. Clarification of phylogenetic relationships of rice genomes is critical for effective utilization of the wild rice germ plasm. By generating and comparing two nuclear gene (Adh1 and Adh2) trees and a chloroplast gene (matK) tree of all rice species, phylogenetic relationships among the rice genomes were inferred. Origins of the allotetraploid species, which constitute more than one-third of rice species diversity, were reconstructed based on the Adh gene phylogenies. Genome types of the maternal parents of allotetraploid species were determined based on the matK gene tree. The phylogenetic reconstruction largely supports the previous recognition of rice genomes. It further revealed that the EE genome species is most closely related to the DD genome progenitor that gave rise to the CCDD genome. Three species of the CCDD genome may have originated through a single hybridization event, and their maternal parent had the CC genome. The BBCC genome species had different origins, and their maternal parents had either a BB or CC genome. An additional genome type, HHKK, was recognized for Oryza schlechteri and Porteresia coarctata, suggesting that P. coarctata is an Oryza species. The AA genome lineage, which contains cultivated rice, is a recently diverged and rapidly radiated lineage within the rice genus.

Metal-organic framework-5 (MOF-5) of composition Zn4O(BDC)3 (BDC = 1,4-benzenedicarboxylate) with a cubic three-dimensional extended porous structure adsorbed hydrogen up to 4.5 weight percent (17.2 hydrogen molecules per formula unit) at 78 kelvin and 1.0 weight percent at room temperature and pressure of 20 bar. Inelastic neutron scattering spectroscopy of the rotational transitions of the adsorbed hydrogen molecules indicates the presence of two well-defined binding sites (termed I and II), which we associate with hydrogen binding to zinc and the BDC linker, respectively. Preliminary studies on topologically similar isoreticular metal-organic framework-6 and -8 (IRMOF-6 and -8) having cyclobutylbenzene and naphthalene linkers, respectively, gave approximately double and quadruple (2.0 weight percent) the uptake found for MOF-5 at room temperature and 10 bar.

OBJECTIVE

Some mothers have difficulty initiating lactation even when highly motivated to breastfeed. The purpose of this study was to determine the incidence of and risk factors for suboptimal infant breastfeeding behavior (SIBB), delayed onset of lactation, and excess neonatal weight loss among mother-infant pairs in a population with high educational levels and motivation to breastfeed.

METHODS

All mothers residing in Davis, California, who gave birth to a healthy, single, term infant at 1 of 5 area hospitals during the 10-month recruitment period in 1999 were invited to participate if they were willing to attempt to breastfeed exclusively for at least 1 month. Lactation guidance was provided and data were collected in the hospital (day 0) and on days 3, 5, 7, and 14. Infant breastfeeding behavior was evaluated by trained lactation consultants using the Infant Breastfeeding Assessment Tool. Onset of lactation was defined based on maternal report of changes in breast fullness. Infant weight loss was considered excessive if it was>>or=10% of birth weight by day 3.

RESULTS

Of the 328 eligible mothers, 280 (85%) participated in the study. The prevalence of SIBB was 49% on day 0, 22% on day 3, and 14% on day 7. SIBB was significantly associated with primiparity (days 0 and 3), cesarean section (in multiparas, day 0), flat or inverted nipples, infant status at birth (days 0 and 3), use of nonbreast milk fluids in the first 48 hours (days 3 and 7), pacifier use (day 3), stage II labor >1 hour (day 7), maternal body mass index >27 kg/m(2) (day 7) and birth weight <3600 g (day 7). Delayed onset of lactation (>72 hours) occurred in 22% of women and was associated with primiparity, cesarean section, stage II labor >1 hour, maternal body mass index >27 kg/m(2), flat or inverted nipples, and birth weight >3600 g (in primiparas). Excess weight loss occurred in 12% of infants and was associated with primiparity, long duration of labor, use of labor medications (in multiparas), and infant status at birth. The risk of excess infant weight loss was 7.1 times greater if the mother had delayed onset of lactation, and 2.6 times greater if the infant had SIBB on day 0.

CONCLUSIONS

Early lactation success is strongly influenced by parity, but may also be affected by potentially modifiable factors such as delivery mode, duration of labor, labor medications, use of nonbreast milk fluids and/or pacifiers, and maternal overweight. All breastfeeding mother-infant pairs should be evaluated at 72 to 96 hours' postpartum.