Human glyceraldehyde-3-phosphate dehydrogenase: mRNA levels and enzyme activity in developing muscle.

Analysis of human glyceraldehyde-3-phosphate dehydrogenase mRNA revealed that levels in adult skeletal muscle are 12-fold greater per microgram of polyadenylated RNA than in fetal skeletal muscle, whereas in cardiac muscle RNA levels were about equal in fetal and adult tissue. The mRNA levels correlate well with glyceraldehyde 3-phosphate dehydrogenase enzyme activities. There was no evidence for fetus- or tissue-specific forms.

California as a model.

One of every three persons who starts smoking falls ill and dies prematurely because he or she smoked. Smoking has been causally linked to heart disease, cancer, and respiratory diseases and continues to be the number one preventable cause of death in this country. To prevent these deaths and the incidence of these diseases, California's Tobacco Control Program was established in 1989 specifically to reduce tobacco use in the state. The strategy of the program is to "denormalize" tobacco. This strategy emphasizes three areas of programmatic activity: to counter pro-tobacco influences, to reduce exposure to environmental tobacco smoke, and to reduce access to tobacco products, with a focus on both social and commercial sources. A fourth priority area, cessation, is considered more of an outcome. California's Tobacco Control Program has touched the life of every Californian. Adult smoking prevalence in the state has gone from approximately 11% lower than the rest of the nation in 1988 to 20% lower in 1996. There are now approximately one million fewer smokers in California than would have been expected. Overall, per capita cigarette consumption has fallen by more than 50%. Seventy percent of adult smokers reported that they tried to quit in the last year. Exposure to secondhand smoke has plummeted. California's lung and bronchus cancer incidence is already declining at a significantly higher rate than that seen elsewhere in the nation. Youth smoking rates have also declined significantly. However, contrary to the message of its massive public relations campaign, the tobacco industry has not changed its stripes after the national tobacco settlement. They are still aggressively marketing their products to teenagers, ethnic minority groups, and young adults. They need to be combatted with renewed vigor by a vigilant health community.

Fine-needle aspiration biopsy in ampullary and pancreatic carcinoma.

Cytological verification of the angiographic, sonographic, and/or intraoperative diagnosis of pancreatic or ampullary carcinoma may be achieved by fine-needle aspiration biopsy (FNAB). Fifteen biopsies were done in 13 patients, 12 of whom were thought to have pancreatic carcinoma clinically. The FNAB specimen confirmed this in 11 of them. Also, one equivocal (suspicious), two false-negative, and no false-positive results were recorded. Complications were not seen. In addition, four patients with ampullary carcinoma had pancreatoduodenectomy on the basis of FNAB specimens. Since this method is more accurate and is safer and simpler than the traditional wedge or core biopsy of suspected pancreatic or ampullary tumors, and since it may eliminate the need for laparotomy in selected cases, we consider it the preferred method for immediate diagnosis of these neoplasms.

Changes of knowledge, attitudes, beliefs, and preference of bar owner and staff in response to a smoke-free bar law.

OBJECTIVE: To examine attitudinal changes of bar owners and staff regarding a smoke-free bar law. DESIGN: Bar owners and staff were random selected and telephone interviewed in June 1998, shortly after a smoke-free bar law was enacted, and October 2002. Similar instruments were used in both surveys to collect data on attitudes related to secondhand smoke (SHS) and behaviours related to the smoke-free bar law. PARTICIPANTS: 651 and 650 respondents worked for either stand alone bars or combination bars. MEASURES: Preference of working in a smoke-free environment, concerns of the effect of SHS, and how to comply with the law. RESULTS: The percentage of bar owners or staff working in stand alone bars who prefer to work in a smoke-free environment increased from 17.3% in 1998 to 50.9% in 2002 (p < 0.001). Significantly more respondents (45.5%) working in stand alone bars were concerned about the effects of SHS on their health, comparing to 21.6% in 1998 (p < 0.001). When patrons smoked in the bar, 82.1% of stand alone bar owners or staff in the 2002 survey would ask them to stop or to smoke outside, increased from only 43.0% in the 1998 survey (p < 0.001). CONCLUSION: A positive and significant attitudinal change related to the smoke-free bar law occurred among California bars.

Disseminated prothothecosis in a dog.

This is a report on the clinical history and pathological lesions of a dog suffering from disseminated protothecosis due to Prototheca zopfi. Clinically, the dog was presented with bilateral conjunctivitis followed by blindness, deafness and posterior paresis. Pathological lesions were most severe in the eyes and consisted of subacute panophthalmitis with secondary posterior subcapsular cataract, posterior synechia, retinal detachment and microscopic evidence of glaucoma. The kidney, liver, brain, spleen and lungs were also affected. This is believed to be the first published account of protothecosis in mammals other than man in Africa. A review of the literature is included.

Fish oil slows prostate cancer xenograft growth relative to other dietary fats and is associated with decreased mitochondrial and insulin pathway gene expression.

BACKGROUND: Previous mouse studies suggest that decreasing dietary fat content can slow prostate cancer (PCa) growth. To our knowledge, no study has yet compared the effect of multiple different fats on PCa progression. We sought to systematically compare the effect of fish oil, olive oil, corn oil and animal fat on PCa progression. METHODS: A total of 96 male severe combined immunodeficient mice were injected with LAPC-4 human PCa cells. Two weeks following injection, mice were randomized to a Western diet based on fish oil, olive oil, corn oil or animal fat (35% kilocalories from fat). Animals were euthanized when tumor volumes reached 1000 mm(3). Serum was collected at death and assayed for PSA, insulin, insulin-like growth factor-1 (IGF-1), IGF-1-binding protein-3 and prostaglandin E-2 (PGE-2) levels. Tumors were also assayed for PGE-2 and cyclooxygenase-2 levels, and global gene expression was analyzed using Affymetrix microarrays. RESULTS: Mice weights and tumor volumes were equivalent across groups at randomization. Overall, fish oil consumption was associated with improved survival relative to other dietary groups (P=0.014). On gene expression analyses, the fish oil group had decreased signal in pathways related to mitochondrial physiology and insulin synthesis/secretion. CONCLUSIONS: In this xenograft model, we found that consuming a diet in which fish oil was the only fat source slowed tumor growth and improved survival compared with that in mice consuming diets composed of olive oil, corn oil or animal fat. Although prior studies showed that the amount of fat is important for PCa growth, this study suggests that the type of dietary fat consumed may also be important.

Effect of intermittent fasting on prostate cancer tumor growth in a mouse model.

Caloric restriction (CR) has been shown to have anti-cancer properties. However, CR may be difficult to apply in humans secondary to compliance and potentially deleterious effects. An alternative is intermittent CR, or in the extreme case intermittent fasting (IF). In a previous small pilot study, we found 2 days per week of IF with ad libitum feeding on the other days resulted in trends toward prolonged survival of mice bearing prostate cancer xenografts. We sought to confirm these findings in a larger study. A total of 100 (7- to 8-week-old) male severe combined immunodeficiency mice were injected subcutaneously with 1 × 10(5) LAPC-4 prostate cancer cells. Mice were randomized to either ad libitum Western Diet (44% carbohydrates, 40% fat and 16% protein) or ad libitum Western Diet with twice-weekly 24 h fasts (IF). Tumor volumes and mouse bodyweights were measured twice weekly. Mice were killed when tumor volumes reached 1000 mm(3). Serum and tumor were collected for analysis of the insulin/insulin-like growth factor 1 (IGF-1) hormonal axis. Overall, there was no difference in mouse survival (P=0.37) or tumor volumes (P ≥ 0.10) between groups. Mouse body weights were similar between arms (P=0.84). IF mice had significantly higher serum IGF-1 levels and IGF-1/IGFBP-3 ratios at killing (P<0.001). However, no difference was observed in serum insulin, IGFBP-3 or tumor phospho-Akt levels (P ≥ 0.39). IF did not improve mouse survival nor did it delay prostate tumor growth. This may be secondary to metabolic adaptations to the 24 h fasting periods. Future studies are required to optimize CR for application in humans.

Preoperative predictors of blood loss at the time of radical prostatectomy: results from the SEARCH database.

The literature contains conflicting data on preoperative predictors of estimated blood loss (EBL) at radical retropubic prostatectomy (RRP). We sought to examine preoperative predictors of EBL at the time of RRP among patients from the SEARCH database to lend clarity to this issue. A total of 1154 patients were identified in the SEARCH database who underwent RRP between 1988 and 2008 and had EBL data available. We examined multiple preoperative factors for their ability to predict EBL using multivariate linear regression analysis. Median EBL was 900 ml (s.d. 1032). The 25th and 75th percentile for EBL were 600 and 1500 ml, respectively. EBL increased significantly with increasing body mass index (BMI) and increasing prostate size and decreased with more recent year of RRP (all P<0.001). The mean-adjusted EBL in normal-weight men (BMI<25 kg/m(2)) was 807 ml compared to 1067 ml among severely obese men (BM I>or=35 kg/m(2)). Predicted EBL for men with the smallest prostates (<20 g) was 721 ml, compared to 1326 ml for men with prostates >or=100 g. Finally, statistically significant differences between centers were observed, with mean-adjusted EBL ranging from 844 to 1094 ml. Both BMI and prostate size are predictors of increased EBL. Prostate size is of particular note, as a nearly twofold increased EBL was seen from the smallest (<20 g) to the largest prostates (>or=100 g). Over time, average EBL significantly decreased. Finally, significant differences in EBL were observed between centers. Patients with multiple risk factors should be forewarned they are at increased risk for higher EBL, which may translate into a greater need for blood transfusion.

Decreased SBPase activity alters growth and development in transgenic tobacco plants.

The effects of reduced SBPase activity on growth and development were examined in a set of transgenic tobacco plants produced using an antisense construct driven by the ribulose bisphosphate carboxylase, small subunit promoter. Photosynthetic carbon assimilation rates and carbohydrate levels in source leaves were decreased in the antisense plants. Growth rate and total shoot biomass were reduced in the SBPase antisense plants, even in plants where SBPase activity was reduced by only 25%. Floral biomass also decreased in response to reductions in SBPase activity and the onset of flowering was delayed by 5-10 d. This is the first demonstration of a link between reproductive biomass and reductions in Calvin cycle enzyme activity using antisense plants. Furthermore, unexpected changes in the growth and development of the antisense plants were evident. Small reductions in SBPase activity (above 50% wild type) resulted in shorter plants with only a small decrease in stem biomass and specific leaf area. In contrast, plants with larger reductions in SBPase activity had an increase in specific leaf area and attained heights similar to that of the wild-type plants but with a much reduced stem biomass, largely due to a decrease in xylem tissue. This bi-modal response of growth to reductions in SBPase activity has similarities to changes in leaf and stem anatomy and morphology that accompany light acclimation.

Photosynthetic capacity is differentially affected by reductions in sedoheptulose-1,7-bisphosphatase activity during leaf development in transgenic tobacco plants.

The impact of reduced sedoheptulose-1,7-bisphosphatase (SBPase) activity on photosynthetic capacity and carbohydrate status was examined during leaf expansion and maturation in antisense transgenic tobacco (Nicotiana tabacum L. cv Samsun) plants. In wild-type plants, photosynthetic capacity was lowest in young expanding leaves and reached a maximum in the fully expanded, mature leaves. In contrast, the transgenic antisense SBPase plants had the highest photosynthetic rates in the young expanding leaves and lowest rates in the mature leaves. In the mature, fully expanded leaves of the transgenic plants photosynthetic capacity was closely correlated with the level of SBPase activity. However, in the youngest leaves of the SBPase antisense plants, photosynthetic rates were close to, or higher than, those observed in wild-type plants, despite having a lower SBPase activity than the equivalent wild-type leaves. Reductions in SBPase activity affected carbohydrate levels in both the mature and young developing leaves. The overall trend was for decreased SBPase activity to lead to reductions in carbohydrate levels, particularly in starch. However, these changes in carbohydrate content were also dependent on the developmental status of the leaf. For example, in young expanding leaves of plants with the smallest reductions in SBPase activity, the levels of starch were higher than in wild-type plants. These data suggest that the source status of the mature leaves is an important determinant of photosynthetic development.

Molecular cloning of the Arabidopsis thaliana sedoheptulose-1,7-biphosphatase gene and expression studies in wheat and Arabidopsis thaliana.

We report here the isolation and nucleotide sequence of genomic clones encoding the chloroplast enzyme sedoheptulose-1,7-bisphosphatase (SBPase) from Arabidopsis thaliana. The coding region of this gene contains eight exons (72-76 bp) and seven introns (75-91 bp) and encodes a polypeptide of 393 amino acids. Unusually, the 5' non-coding region contains two additional AUG codons upstream of the translation initiation codon. A comparison of the deduced Arabidopsis and wheat SBPase polypeptide sequences reveals 78.6%, identity. Expression studies showed that the level of SBPase mRNA in Arabidopsis and wheat is regulated in a light-dependent manner and is also influenced by the developmental stage of the leaf. Although the Arabidopsis SBPase gene is present in a single copy, two hybridizing transcripts were detected in some tissues, suggesting the presence of alternate transcription start sites in the upstream region.

Molecular biology of the C3 photosynthetic carbon reduction cycle.

In recent years the enzymes of the C3 photosynthetic carbon reduction (PCR) cycle have been studied using the techniques of molecular biology. In this review we discuss the primary protein sequences and structural predictions that have been made for a number of these enzymes, which, with the input of crystallographic analysis, gives the opportunity to understand the mechanisms of enzyme activity.The genome organisation and gene structure of the PCR enzymes is another area which has recently expanded, and we discuss the regulation of the genes encoding these enzymes and the complex interaction of various factors which influence their expression.

pho3: a phosphorus-deficient mutant of Arabidopsis thaliana (L.) Heynh.

A novel P-deficient mutant of Arabidopsis thaliana, pho3, was isolated by screening for root acid phosphatase (APase) activity in plants grown under low-P conditions. pho3 had 30% less APase activity in roots than the wild type and, in contrast to wild-type plants, root APase activity did not increase in response to growth in low P. However, shoot APase activity was higher in pho3 than in the wild-type plants. In addition, the pho3 mutant had a P-deficient phenotype, even when grown in P-sufficient conditions. The total P content of 11-d-old pho3 plants, grown in agar media with a plentiful supply of P, was about 25% lower than the wild-type level in the shoot, and about 65% lower in the roots. In the rosette leaves of mature soil-grown pho3 plants the total P content was again reduced, to about 50% of wild-type levels. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased anthocyanin content (at least 100-fold greater than the wild type in soil-grown plants) and starch accumulation. The results suggest that the mutant is unable to respond to low internal P levels, and may lack a transporter or a signalling component involved in regulating P nutrition.

Chloroplast fructose-1,6-bisphosphatase: the product of a mosaic gene.

We show here that light stimulates the expression of nuclear genes in wheat leaves for chloroplast fructose-1,6-bisphosphatase (FBPase) and describe a sequence of amino acids in this enzyme which may be responsible, via thioredoxin, for the light regulation of its activity. This data results from (a) our isolation and characterization of a cDNA of this enzyme which contains its entire coding sequence, and (b) our use of this cDNA as a probe to detect mRNA levels in wheat plants subjected to different light regimes. The similarity in amino acid sequence of the encoded enzyme from diverse sources suggests that the FBPase genes all had a common origin. However, their control sequences have been adjusted so that they are appropriately expressed and their coding sequences modified so that the enzymic activity of their products are suitably regulated in the particular cellular environment in which they must function. The light-activated regulatory sequences in the gene for the chloroplast protein have probably come together by a shuffling of DNA segments.

Complete coding sequence of wheat phosphoribulokinase: developmental and light-dependent expression of the mRNA.

We have isolated and sequenced a cDNA clone which contains the entire coding sequence of the precursor to a subunit of wheat phosphoribulokinase (PRKase). (The enzyme is a homodimer). The cDNA contains 1533 bp and has an open reading frame of 1212 nucleotides. This encodes a protein with an amino-terminal transit sequence of 53 amino acids, while the part that forms the mature protein contains 351 amino acids and has a molecular weight of 39,200 daltons. A comparison of the wheat amino acid sequence with that already known for the mature protein of spinach reveals that there are identical residues in 86% of the positions but their transit peptides differ substantially from one another. The mature wheat and spinach proteins are identical in a segment of over 50 amino acids near the amino-terminus which is the region believed to be involved in ATP binding and in regulation by light of the catalytic activity of the enzyme. We further demonstrate that the expression of PRKase mRNA in wheat leaves is regulated in a developmental, tissue-specific and light dependent manner. We also show that the light-induced increase in the steady-state levels of this mRNA is dependent on the developmental stage of the leaf.

cDNA and gene sequences of wheat chloroplast sedoheptulose-1,7-bisphosphatase reveal homology with fructose-1,6-bisphosphatases.

The nucleotide sequence encoding the chloroplast enzyme, sedoheptulose-1,7-bisphosphatase [Sed(1,7)P2ase], was obtained from wheat cDNA and genomic clones. The transcribed region of the Sed(1,7)P2ase gene has eight exons (72-507 bp) and seven introns (85-626 bp) and encodes a precursor polypeptide of 393 amino acids. Comparison of the deduced amino acid sequence of Sed(1,7)P2ase with those of fructose-1,6-bisphosphatase [Fru(1,6)P2ase] enzymes from a variety of sources reveals 19% identity, rising to 42% if conservative changes are considered. Most importantly, the amino acid residues which form the active site of Fru(1,6)P2ase are highly conserved in the Sed(1,7)P2ase molecule, indicating a common catalytic mechanism. Interestingly, although the activities of both Sed(1,7)P2ase and chloroplast Fru(1,6)P2ase are modulated by light via the thioredoxin system, the amino acid sequence motif identified as having a role in this regulation in chloroplast Fru(1,6)P2ase is not found in the Sed(1,7)P2ase enzyme.

Arabidopsis thaliana carbonic anhydrase: cDNA sequence and effect of CO2 on mRNA levels.

A full-length cDNA clone encoding carbonic anhydrase was isolated from an Arabidopsis thaliana (Columbia) leaf library. Comparison of the derived amino acid sequence obtained from this clone with those of pea and spinach reveals a considerable degree of identity. The carbonic anhydrase cDNA was used to probe the level of RNA encoding this protein in the leaves of plants grown in elevated CO2 (660 ppm). We have found that under these conditions the steady-state level of carbonic anhydrase mRNA was increased in comparison with control plants grown in normal atmospheric concentrations of CO2 (330 ppm). This raises the intriguing possibility that there exists in higher plants a mechanism for perceiving and responding to changes in environmental CO2 concentrations at the genetic level.

The chloroplast FBPase gene of wheat: structure and expression of the promoter in photosynthetic and meristematic cells of transgenic tobacco plants.

A gene encoding chloroplast fructose-1,6-bisphosphatase (FBPase) was isolated from a genomic library of wheat DNA. Comparison of the gene sequence obtained with that of a wheat cDNA clone revealed the presence of three introns, each less than 100 bases in length. One of these introns lies in a region that may be involved in the light activation of FBPase catalytic activity. Chimeric gene constructs comprising 1673 bp of the upstream FBPase promoter region in a transcriptional fusion to the beta-glucuronidase (GUS) reporter gene were used to investigate expression in transgenic tobacco plants. Histochemical localization of GUS activity revealed high levels of expression driven by the FBPase promoter sequences in photosynthetically active tissues and, unexpectedly, also in the meristematic regions of shoots, lateral buds and roots. The biological significance of FBPase expression in meristematic regions is not yet clear but this pattern of expression may be explained by the presence in the FBPase promoter of a short DNA sequence motif which is also found in the CaMV 35S viral promoter.