Cloning of the y1 Locus of Maize, a Gene Involved in the Biosynthesis of Carotenoids.

The y1 gene is one of the genes responsible for the production of [beta]-carotene in the endosperm and leaves of maize. We have cloned a Robertson's Mutator-tagged allele of the y1 gene (y1-mum) by using a Mu3 element as a hybridization probe. We substantiate that the cloned sequence is a portion of the y1 gene by molecular analyses of a revertant of a putative Mutator-induced y1 allele and the incidence of insertions within the cloned y1 sequence from several independently derived Mutator-induced y1 mutant stocks. The y1-mum sequence was used to isolate the standard Y1 allele, which conditions the presence of [beta]-carotene in the endosperm of the maize kernel.

The y1 gene of maize codes for phytoene synthase.

The cloned y1 locus of maize was sequenced and found to encode phytoene synthase. Different "wild-type" alleles of the locus were found to differ by the insertion of transposable elements in their promoter and polyA addition regions, and by the length of a CCA tandem repeat series, without any obvious effect on function of the gene. A dominant Y1 ("wild-type") allele was observed to be expressed at highest levels in the seedling but also in the embryo and endosperm. The Mu3 transposable element insertion responsible for a pastel allele of y1, which gives lowered levels of carotenoids in the endosperm of kernels and seedlings grown at high temperatures, was located in the 5' end of the gene. Although the size of the transcript from this y1 mutation suggests that the Mu3 element provides the promoter for this allele, leaf tissue in this mutant line contained approximately normal amounts of y1 mRNA. A recessive allele of y1, which conditions normal levels of carotenoids in the embryo and seedling, but almost no carotenoids in the endosperm, was found to accumulate normal amounts of y1 mRNA in the seedling and embryo, while y1 transcripts were not detected in the endosperm.

Optimized determination of angiotensin I-converting enzyme activity with hippuryl-L-histidyl-L-leucine as substrate.

The determination of the angiotensin I-converting enzyme activity (ACE, kininase II, peptidyldipeptide hydrolase, EC 3.4.15.1) is necessary to control the course and the treatment of sarcoidosis, as well as to monitor the therapeutic use of enzyme inhibitors such as captopril in hypertension or congestive heart failure. Numerous synthetic substrates are known with which to measure the enzyme activity. A discontinuous method using hippuryl-L-histidyl-L-leucine was tested and improved. The cleavage product, hippurate, reacts with cyanuric chloride to give a yellow complex which can be measured at 405 nm using a spectral line photometer. Enzyme activity, kinetic constants and activation energy are dependent on the chloride ion concentration. Optimal test concentrations are 1.1 mol/l potassium chloride and 3.0 mmol/l hippuryl-L-histidyl-L-leucine at pH 8.3. Higher substrate concentrations effect an inhibition of the enzyme reaction. A Michaelis constant of 0.9 mmol/l was found with serum as enzyme source. An activation energy of 57 kJ/mol was obtained from the relation between the logarithm of velocity of enzyme reaction and reciprocal value of absolute temperature. Furthermore, a linear dependence on chloride ion concentration was observed. The histogram of the enzyme activities in sera from 146 healthy volunteers shows a non-gaussian distribution. The reference interval at 25 degrees C is characterized by a median of 24 units/l with the 2.5th and the 97.5th percentiles at 13 units/l and 42 units/l, respectively. The corresponding values at 37 degrees C are 27 units/l and 86 units/l with a median of 48 units/l. No significant sex and age dependence could be found. A potent ACE inhibitor such as captopril leads to a rapid decrease of the enzyme activity within 60 min after oral administration. In the following hours, the enzyme activity slowly increases.

Microbiological assessment of an improved stannous fluoride dentifrice.

Recently, a new stabilized stannous fluoride (SnF2) dentifrice (SSF) has been developed. The aim of the present work was to examine the antimicrobial activity of SnF2, and to assess the long-term microbial safety of this dentifrice in a series of in vitro and clinical evaluations. Results of in vitro time-kill experiments with representative oral bacteria demonstrated that SnF2 exerts broad antimicrobial activity against both Gram-positives and Gram-negatives and, in particular, has potent activity against Streptococcus mutans. Sixty-eight subjects participated in a nine-day plaque regrowth clinical study to assess the short-term antiplaque effect of SSF. The results revealed no significant differences from the negative control, suggesting that SnF2 does not detectably or directly alter plaque microbial viability or composition. Separately, evaluation of microbial safety in a subgroup of 120 subjects participating in a six-month clinical efficacy and safety trial found no significant ecological shifts between SSF and the negative (NaF dentifrice) control among 11 supragingival microbial populations examined. The potential for development of bacterial resistance to SnF2 was assessed under both in vitro and clinical conditions. In a rigorous assessment of the ability of bacterial populations to develop either phenotypic or genotypic resistance to SnF2, representative bacteria were exposed to continuous sub-lethal concentrations of SnF2 in a laboratory chemostat for at least 9 days. Results of time-kill experiments on exposed populations revealed no significant changes in susceptibility despite exposure of over 10(12) bacteria. Based on typical spontaneous mutation rates of 10(-6) to 10(-8), these results suggested that the potential for bacteria to develop resistance to SnF2 is low. Evaluation of susceptibility to SnF2 to over 800 bacterial isolates obtained over the course of the six-month clinical trial corroborate the in vitro findings, revealing no changes in susceptibility suggestive of development of resistance to SnF2 is a microbiologically safe agent for oral use and support separate clinical observations demonstrating the safety and efficacy of this stabilized SnF2 dentifrice.

Age, poverty and alcohol use as HIV risk factors for women in Mongu, Zambia.

BACKGROUND: Age, poverty and alcohol use are seen as risk factors for HIV among women in sub-Saharan Africa. OBJECTIVE: The objective of this study was to understand the influence of socioeconomic factors (including age and poverty) as well as alcohol use on risky sexual behaviors among women in Mongu, Zambia. METHODS: This study examines these factors in the local context of Mongu, Zambia using the Priorities for Local AIDS Control Efforts (PLACE) methodology. This methodology allows for the study of risky behaviors while taking into consideration local factors. The two outcome variable studied were transactional sex in the past year and having two or more sexual partners in the past year. RESULTS: In this study age was not a significant factor, but alcohol use and poverty/desire for economic advancement were significant factors. CONCLUSION: Programs and policies need to address the influence of alcohol on risky sexual behaviors and also the important but complex influence of poverty.

Age; Poverty and Alcohol Use as HIV Risk Factors for Women in Mongu; Zambia

Background: Age; poverty and alcohol use are seen as risk factors for HIV among women in sub-Saharan Africa. Objective: The objective of this study was to understand the influence of socioeconomic factors (including age and poverty) as well as alcohol use on risky sexual behaviors among women in Mongu; Zambia. Methods: This study examines these factors in the local context of Mongu; Zambia using the Priorities for Local AIDS Control Efforts (PLACE) methodology. This methodology allows for the study of risky behaviors while taking into consideration local factors. The two outcome variable studied were transactional sex in the past year and having two or more sexual partners in the past year. Results: In this study age was not a significant factor; but alcohol use and poverty/desire for economic advancement were significant factors. Conclusion: Programs and policies need to address the influence of alcohol on risky sexual behaviors and also the important but complex influence of poverty

Monitoring trends in sexual behaviour in Zambia, 1996-2003.

OBJECTIVES: This paper assesses the evidence of changes in sexual behaviour in Zambia, accounting for differences in the composition of survey samples and for evidence of changes in reporting bias. It compares the estimates of sexual behaviour measures obtained using two different survey methodologies. METHODS: Data from five nationally representative household surveys carried out between 1996 and 2003 were analysed for change in selected sexual behaviours. RESULTS: There is some evidence for an increase in men's age at first sex. The proportion of people reporting multiple partnerships and not using a condom at last sex has declined since 1996. Unprotected sex with non-cohabiting partners is reported less frequently in the later surveys. The socioeconomic and demographic composition of the survey samples has changed across the years but the declines in behaviour remain statistically significant after adjustment for these changes. There is evidence of a changing reporting bias over the period of interest, with respondents less likely to report a young age at first sex in later surveys. CONCLUSIONS: Between 1996 and 2003, reported sexual behaviour has changed in Zambia among both men and women. Different survey methods produce significantly different estimates of sexual behaviour. This must be taken into account when carrying out trend analyses using data from different sources.

Chloroplast DNA variation between the common cultivated potato (Solanum tuberosum ssp. tuberosum) and several South American relatives.

Chloroplast DNA (ctDNA) from the tuberbearing Solanum species tuberosum, vernei, phureja, and chacoense has been compared by restriction endonuclease analysis. Digestion by Hind III or Xba I reveal no differences, but digestion with Bam HI and Eco RI reveals minor differences in the ctDNA among these species. The ctDNA restriction patterns of the tetraploid common cultivated potato of North America and Europe, S. tuberosum ssp. tuberosum and the South American tetraploid, S. tuberosum ssp. andigena are identical for all four restriction endonucleases. These data suggest that ssp. tuberosum and ssp. andigena contain similar ctDNA and therefore may share a common ancestor, or direct lineage. The ctDNA restriction patterns of S. vernei and S. chacoense are identical for all four restriction endonucleases, and S. phureja ctDNA, can be distinguished from the other diploid ctDNAs by digestion with Bam HI. None of the diploids analyzed contain ctDNA identical to the tetraploids and therefore either did not contribute their chloroplast genomes to the evolution of the tetraploids, or the ctDNA has diverged since this evolutionary event. The ctDNAs studied did not contain restriction polymorphisms which could be correlated to cytoplasmic male sterility in Solanum. This is the first demonstration of ctDNA diversity in the tuber-bearing Solanum species.

Butyrate and propionate: important components of toxic dental plaque extracts.

Extracts of in vitro-cultured human dental plaque contain factors toxic to mammalian cells. Previous studies demonstrated that those toxic factors most readily released from cultured plaque had very low molecular weights and were heat stable. Studies reported here demonstrate that metabolic end products including short-chain fatty acids were present in fractions containing the low-molecular-weight, heat-stable factors. The salts of two of these acids, butyrate and propionate, inhibited proliferation of both mouse L929 cells and human gingival fibroblasts. Furthermore, when tested at concentrations present in plaque extracts, the inhibitory effects of butyrate and propionate accounted for essentially all the inhibitory potential of the extracts. These findings, taken together with those of other groups, suggest that butyrate and propionate, end products of dental plaque metabolism, may have an etiological role in periodontal disease.

Impact of IPAs on fee-for-service medical groups.

Individual practice association (IPA) is the newest and most rapidly growing HMO model. Despite potential benefits, IPAs have experienced a variety of difficulties. A case study analysis of community practices in North Carolina revealed satisfactory results as well as concerns.

Organization of the ribosomal RNA genes of Schizophyllum commune.

The 18, 5.8, 25 and 5S ribosomal RNA (rRNA) cistrons have been mapped on the ribosomal DNA (rDNA) unit repeat of Schizophyllum commune strain 4-40. These genes are spatially ordered in the sequence given. The presence of a large primary precursor rRNA which is processed to form the mature 18, 5.8 and 25S rRNAs has been demonstrated. We have mapped the site of transcriptional initiation for this rRNA primary precursor. The sequence surrounding this site has been determined and shown to be highly conserved, with considerable identity to those in Neurospora crassa and Dictyostelium discoideum. The direction of transcription of the rRNA genes has been determined. The 5S rRNA cistron is transcribed in the same direction as the other rRNAs, however it is not transcribed as a part of the large primary precursor. The previously identified rDNA strain-specific length polymorphisms (Specht et al. 1984) are shown to be located within the transcribed region of the rDNA unit repeat.

Microsatellite repeat variation within the y1 gene of maize and teosinte.

We have sequenced a (CCA)n microsatellite-containing region of the y1 gene from 11 different lines of maize and 6 teosinte species, subspecies, or varieties. The (CCA)n microsatellite was found to vary in repeat number from 3 to 11. In addition, a pentanucleotide repeat adjacent to the trinucleotide microsatellite exhibits sequence and repeat number variation. Therefore, the (CCA)n microsatellite, as well as the sequence directly adjacent to it, exhibit variability in both maize and teosinte and could potentially serve as molecular markers in mapping or breeding studies.

Hemorrhagic adrenal metastases: CT findings.

Adrenal hemorrhage secondary to metastases is uncommon. We have encountered four such cases that presented as large adrenal masses. In all cases the CT findings were of an inhomogeneous mixed-density adrenal mass with extensive perirenal changes suggestive of perirenal hemorrhage or mass. When such a lesion is seen, hemorrhagic adrenal metastases should be considered. Fluid may be of high density, suggestive of hemorrhage. However, as the adrenal is in the perinephric space, hemorrhage from any cause (trauma, metastases, or anticoagulant) in the adrenal gland will gravitate into the perinephric space.

Fibroproliferation and mast cells in the acute respiratory distress syndrome.

BACKGROUND: Mast cells (MCs), which are a major source of cytokines and growth factors, have been implicated in various fibrotic disorders. To clarify the contribution of MCs to fibrogenesis, lung tissue from patients with the acute respiratory distress syndrome (ARDS) was examined during exudative through to fibroproliferative stages. METHODS: Lung tissue was obtained from 17 patients with ARDS who had pathological features of the early exudative stage (n = 6) or the later reparative stages (n = 11), from four patients with idiopathic pulmonary fibrosis, and from three patients with normal lung tissue. Immunohistochemical localisation of tryptase (found in all human MCs), chymase (found in a subset of human MCs), alpha-smooth muscle actin (identifies myofibroblasts), and procollagen type I was performed. RESULTS: Normal lung tissue exhibited myofibroblast and procollagen type I immunolocalisation scores each of < 5 and MC scores of 1. Increased scores were defined as myofibroblast and procollagen type I scores of > 10 and MC scores of > or = 2. Eighty percent of lung tissue samples from the early exudative stage of ARDS exhibited increased numbers of myofibroblasts, 50% had increased numbers of procollagen type I producing cells, while only 17% had increased numbers of MCs compared with control samples. All samples from the later reparative stages of ARDS had increased numbers of myofibroblasts and procollagen type I producing cells. Increased numbers of MCs were seen in 55% of samples from the reparative stages. There was no significant shift in MC phenotype in the ARDS samples. CONCLUSIONS: Increased numbers of myofibroblasts and procollagen type I producing cells were frequently found early in the course of ARDS. MC hyperplasia was unusual during this stage, but was often a feature of the later reparative stages. MCs do not appear to initiate fibroproliferation in ARDS.