Mixed-power scaling of whole-plant respiration from seedlings to giant trees.

The scaling of respiratory metabolism with body mass is one of the most pervasive phenomena in biology. Using a single allometric equation to characterize empirical scaling relationships and to evaluate alternative hypotheses about mechanisms has been controversial. We developed a method to directly measure respiration of 271 whole plants, spanning nine orders of magnitude in body mass, from small seedlings to large trees, and from tropical to boreal ecosystems. Our measurements include the roots, which have often been ignored. Rather than a single power-law relationship, our data are fit by a biphasic, mixed-power function. The allometric exponent varies continuously from 1 in the smallest plants to 3/4 in larger saplings and trees. Therefore, our findings support the recent findings of Reich et al. [Reich PB, Tjoelker MG, Machado JL, Oleksyn J (2006) Universal scaling of respiratory metabolism, size, and nitrogen in plants. Nature 439:457-461] and West, Brown, and Enquist [West GB, Brown JH, Enquist BJ (1997) A general model for the origin of allometric scaling laws in biology. Science 276:122 -126.]. The transition from linear to 3/4-power scaling may indicate fundamental physical and physiological constraints on the allocation of plant biomass between photosynthetic and nonphotosynthetic organs over the course of ontogenetic plant growth.

DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1.

About 90 percent of human pancreatic carcinomas show allelic loss at chromosome 18q. To identify candidate tumor suppressor genes on 18q, a panel of pancreatic carcinomas were analyzed for convergent sites of homozygous deletion. Twenty-five of 84 tumors had homozygous deletions at 18q21.1, a site that excludes DCC (a candidate suppressor gene for colorectal cancer) and includes DPC4, a gene similar in sequence to a Drosophila melanogaster gene (Mad) implicated in a transforming growth factor-beta (TGF-beta)-like signaling pathway. Potentially inactivating mutations in DPC4 were identified in six of 27 pancreatic carcinomas that did not have homozygous deletions at 18q21.1. These results identify DPC4 as a candidate tumor suppressor gene whose inactivation may play a role in pancreatic and possibly other human cancers.

Household solid waste characteristics and management in Chittagong, Bangladesh.

Solid waste management (SWM) is a multidimensional challenge faced by urban authorities, especially in developing countries like Bangladesh. We investigated per capita waste generation by residents, its composition, and the households' attitudes towards waste management at Rahman Nagar Residential Area, Chittagong, Bangladesh. The study involved a structured questionnaire and encompassed 75 households from five different socioeconomic groups (SEGs): low (LSEG), lower middle (LMSEG), middle (MSEG), upper middle (UMSEG) and high (HSEG). Wastes, collected from all of the groups of households, were segregated and weighed. Waste generation was 1.3 kg/household/day and 0.25 kg/person/day. Household solid waste (HSW) was comprised of nine categories of wastes with vegetable/food waste being the largest component (62%). Vegetable/food waste generation increased from the HSEG (47%) to the LSEG (88%). By weight, 66% of the waste was compostable in nature. The generation of HSW was positively correlated with family size (r xy=0.236, p<0.05), education level (r xy=0.244, p<0.05) and monthly income (r xy=0.671, p<0.01) of the households. Municipal authorities are usually the responsible agencies for solid waste collection and disposal, but the magnitude of the problem is well beyond the ability of any municipal government to tackle. Hence dwellers were found to take the service from the local waste management initiative. Of the respondents, an impressive 44% were willing to pay US dollars 0.3 to US dollars 0.4 per month to waste collectors and it is recommended that service charge be based on the volume of waste generated by households. Almost a quarter (22.7%) of the respondents preferred 12-1 pm as the time period for their waste to be collected. This study adequately shows that household solid waste can be converted from burden to resource through segregation at the source, since people are aware of their role in this direction provided a mechanism to assist them in this pursuit exists and the burden is distributed according to the amount of waste generated.

An integrated high-resolution physical map of the DPC/BRCA2 region at chromosome 13q12.

We identified a homozygous deletion in a pancreatic carcinoma (DPC) that localized to a 1-cM region at chromosome 13q12.3, which lay within the 6-cM locus of familial breast cancer susceptibility (BRCA-2). Here we present a physical map of the region, consisting of YAC, PAC, and cosmid contigs. The YAC contig comprises 16 clones that together span the entire BRCA2 region. The PAC contig comprises 22 clones that together span the DPC region. Seventy cosmid clones were localized within and near the DPC region. Thirty-five sequence-tagged sites were defined and localized within the map. The map indicates the size of the DPC region to be near 250 kb, and provides mapped and cloned resources for the search for the putative tumor suppressor gene(s) in the region.

Allelotype of pancreatic adenocarcinoma using xenograft enrichment.

p53 and MTS1 are known to be mutationally inactivated in pancreatic adenocarcinoma. Other tumor suppressor genes are likely also to play a role. To define chromosomal arms which may harbor additional tumor suppressor genes, we performed an extensive allelotype on pancreatic cancer utilizing a xenograft enrichment technique. Eighty-eight percent (28/32) of primary tumors gave rise to xenografts. Eighteen cases were used in a PCR-based allelotype using 283 polymorphic markers, over 2800 informative assays, and an average coverage of 4.1 informative markers per chromosomal arm per case. Highly frequent allelic loss (> 60%) was seen at chromosomes 1p, 9p, 17p, and 18q. Moderately frequent allelic loss (40-60%) was seen at 3p, 6p, 6q, 8p, 10q, 12q, 13q, 18p, 21q, and 22q. The average fractional allelic loss was 0.36. Allelic and sequence stability was demonstrated among 64 parallel and second-passage xenografts derived from 12 cases of pancreatic adenocarcinoma with the ascertainment of over 3000 single alleles. The findings were confirmed in primary tumors. In only two instances were discrepancies revealed between the allelic loss data obtained from corresponding parallel xenografts, probably due to the xenografting of minor subpopulations, reflecting genetic heterogeneity of the primary tumor.

Homozygous deletion map at 18q21.1 in pancreatic cancer.

Absolute genetic differences between neoplastic and nonneoplastic cells can be discerned at sites of homozygous deletions. These deletions are of critical interest because they might be useful in the identification of defective biochemical pathways in neoplastic cells, and subsequently for the development of new treatment strategies in human cancer. We identified an area at 18q21.1 involved by homozygous deletions in 30% of pancreatic carcinomas. To characterize the homozygous deletions, we constructed a detailed physical map of nearly 2 Mb, containing yeast artificial chromosomes, P1-derived artificial chromosomes, cosmids and 24 sequence-tagged sites. The homozygously deleted are contained a new candidate tumor-suppressor gene (DPC4). To date, 23 (64%) of 35 pancreatic carcinomas carry at least one homozygous deletion at a published locus. The study of the total gene content of these loci, facilitated by the sequence-tagged site markers and maps of these regions, should help to reveal the absolute biochemical differences between neoplastic and nonneoplastic cells for a common human tumor.

Partial redirection of transgenic human growth hormone secretion from rat salivary glands.

Regulated secretory pathway proteins, when delivered as transgenes to salivary glands, are secreted predominantly into saliva. This is not useful for those proteins whose therapeutic function is required systemically, for example, human growth hormone (hGH). One strategy to improve the efficiency of hGH secretion into the bloodstream involves manipulation of existing sorting signals. The C terminus of hGH is highly conserved and contains a domain similar to the regulated pathway sorting domain of pro-opiomelanocortin (POMC). We hypothesized that, similar to POMC, mutation of this domain would divert hGH secretion from the regulated to the constitutive pathway, which in salivary glands leads to the bloodstream. Several mutations were made in the C terminus of the hGH cDNA and tested in vitro. One biologically active mutant containing E174A and E186A substitutions, and with an included C-terminal extension, was studied in greater detail. Compared with wild-type hGH, we found that this mutant hGH accumulated in the Golgi/trans-Golgi network and showed increased basal secretion in AtT20 cells, a model endocrine cell line. Importantly, in vivo, the mutant hGH displayed a relative increase in the proportion of constitutive pathway secretion seen from rat salivary glands, with a significantly lower saliva-versus-serum secretion ratio (p=0.03). Although this mutant is unlikely to be therapeutically beneficial, these results suggest that the final destination of a transgenic secretory protein may be controlled by reengineering its sorting determinants.

Hydroxychloroquine enhances the endocrine secretion of adenovirus-directed growth hormone from rat submandibular glands in vivo.

Use of gene transfer technology for treating single protein deficiency disorders requires delivery of therapeutic levels of the transgene product. We have suggested that salivary glands may provide a potentially valuable target site for certain systemic applications of gene therapeutics (He et al., Gene Ther. 1998;5:537-541). However, the ability of salivary glands to deliver therapeutic proteins to either the upper gastrointestinal tract via saliva or to the bloodstream, as required, must be carefully evaluated. In the anterior pituitary gland, human growth hormone (hGH) is secreted into the bloodstream via the regulated secretory pathway. However, when expressed from an adenoviral vector delivered to salivary glands, most hGH follows the regulated, tissue-specific, exocrine secretory pathway into saliva, where it is not therapeutically useful. We tested the hypothesis that the commonly used, FDA-approved drug hydroxychloroquine (HCQ) can divert adenovirus-directed hGH from this regulated secretory pathway in rat submandibular glands and enhance delivery into the bloodstream. In untreated rats, there was approximately 20-fold more vector-directed hGH in saliva than in serum. Administration of HCQ led to a shift of hGH secretion into the bloodstream. When delivered at doses of 1 or 10 mg/kg body weight, via intraperitoneal injection plus intraductal infusion, the saliva:serum hGH ratio was approximately 2:1. Such HCQ delivery did not significantly alter the total amount of hGH measured, but increased the serum level of hGH 5- to 6-fold. Also, HCQ had no significant effects on serum chemistries or hematological parameters. We conclude that HCQ is able to significantly enhance hGH secretion from salivary glands into the bloodstream and may be useful to facilitate clinical applications of gene therapeutics via salivary glands.

Evaluation of salivary gland acinar and ductal cell-specific promoters in vivo with recombinant adenoviral vectors.

Adenoviral vectors efficiently deliver exogenous genes to salivary glands. There are two general epithelial cell types, with very different functions, in salivary glands--acinar and ductal. To determine if gene expression can be restricted in vivo to either general cell type using a relatively cell/tissue-specific promoter in conjunction with adenovirus-mediated gene transfer, we tested the human amylase and kallikrein promoters. For initial studies the sensitive reporter gene luciferase was used in two adenoviral constructs. The adenovirus AdAMY-luc contains the human salivary gland amylase promoter (-1003 to +2)(AMY1C) and AdKALL-luc contains the human tissue kallikein promoter (-315 to -1)(KLK1). The adenovirus AdKALL-hAQP1 was also used to test a therapeutic gene, human aquaporin-1 (hAQP1), potentially of importance in treating surviving ductal cells in irradiation-damaged glands. Luciferase expression after AdAMY-luc delivery in vivo directly to the parotid, submandibular, and sublingual glands, as well as to the lungs, and intravenously via the femoral vein, was restricted to the three salivary glands and the pancreas. AdKALL-luc delivery via the same routes resulted in a more general distribution of luciferase expression, although greatest luciferase activity was seen in salivary glands and lung. Luciferase activity after AdAMY-luc delivery was proportionally greater (approximately 14-fold) in acinar cells, whereas luciferase activity after AdKALL-luc delivery was proportionally greater (approximately 9-fold) in ductal cells. The expression of hAQP1 after AdKALL-hAQP1 gene transfer was mainly observed in ductal cells in vivo. AdKALL-hAQP1 was as useful as AdCMV-hAQP1 in increasing salivary flow rates of irradiated rats. This study demonstrates that adenoviral vectors containing the relatively cell/tissue-specific AMY1C or KLK1 promoters may be useful for targeting therapeutic gene expression in salivary glands.

Polarized secretion of transgene products from salivary glands in vivo.

Previously (Kagami et al. Hum. Gene Ther. 1996;7:2177-2184) we have shown that salivary glands are able to secrete a transgene-encoded protein into serum as well as saliva. This result and other published data suggest that salivary glands may be a useful target site for vectors encoding therapeutic proteins for systemic delivery. The aim of the present study was to assess in vivo if transgene-encoded secretory proteins follow distinct, polarized sorting pathways as has been shown to occur "classically" in cell biological studies in vitro. Four first-generation, E1-, type 5 recombinant adenoviruses were used to deliver different transgenes to a rat submandibular cell line in vitro or to rat submandibular glands in vivo. Subsequently, the secretory distribution of the encoded proteins was determined. Luciferase, which has no signal peptide, served as a cell-associated, negative control and was used to correct for any nonspecific secretory protein release from cells. The three remaining transgene products tested, human tissue kallikrein (hK1), human growth hormone (hGH), and human alpha1-antitrypsin (halpha1AT), were predominantly secreted (>96%) in vitro. Most importantly, in vivo, after a parasympathomimetic secretory stimulus, both hK1 and hGH were secreted primarily in an exocrine manner into saliva. Conversely, halpha1AT was predominantly secreted into the bloodstream, i.e., in an endocrine manner. The aggregate results are consistent with the recognition of signals encoded within the transgenes that result in specific patterns of polarized protein secretion from rat submandibular gland cells in vivo.

Cochlear gene delivery through an intact round window membrane in mouse.

Cochlear gene transfer studies in animal models have utilized mainly two delivery methods: direct injection through the round window membrane (RWM) or intracochlear infusion through a cochleostomy. However, the surgical trauma, inflammation, and hearing loss associated with these methods lead us to investigate a less invasive delivery method. Herein, we studied the feasibility of a vector transgene-soaked gelatin sponge, Gelfoam, for transgene delivery into the mouse cochlea through an intact RWM. The Gelfoam absorbed with liposomes and adenovirus, but not with adeno-associated virus (AAV), was successful in mediating transgene expression across an intact RWM in a variety of cochlear tissues. The Gelfoam technique proved to be an easy, atraumatic, and effective, but vector-dependent, method of delivering transgenes through an intact RWM. Compared with the more invasive gene delivery methods, this technique represents a safer and a more clinically viable route of cochlear gene delivery in humans.

Construction and function of a recombinant adenovirus encoding a human aquaporin 1-green fluorescent protein fusion product.

Transfer of the human aquaporin 1 (hAQP1) gene provides a novel way to potentially correct the severe salivary hypofunction associated with therapeutic radiation for head and neck cancer. To facilitate the study of individual cells transduced with this gene, we have designed a fusion product of the hAQP1 and jellyfish green fluorescent protein (GFP) cDNAs. An expression plasmid, pACCMVhAQP1GFP, and a recombinant adenovirus, AdhAQP1GFP, encoding this fusion product were constructed. Both the recombinant plasmid and virus directed the expression of the encoded, 55-kDa fusion protein (hAQP1GFP), which was detected in the plasma membranes of several epithelial cell lines (293, SMIE, and A5). hAQP1GFP was functionally active and facilitated fluid movement across a polarized salivary epithelial cell monolayer (approximately 5-fold noninfected controls) in response to an osmotic gradient. In response to a hypotonic challenge, individual epithelial cells expressing the fusion protein exhibited significantly more capacitance (used herein as an indicator of cell swelling) than control cells. Conversely, in response to a hypertonic challenge, individual infected cells shrunk more rapidly (approximately 2- to 3-fold) and to a greater extent than control cells. We conclude that AdhAQP1GFP is a useful experimental tool to identify and study individual cells expressing a water channel transgene.

Safety and efficacy of adenovirus-mediated transfer of the human aquaporin-1 cDNA to irradiated parotid glands of non-human primates.

This study evaluated the safety and efficacy of a single administration of a recombinant adenovirus encoding human aquaporin-1 (AdhAQP1) to the parotid glands of adult rhesus monkeys. In anticipation of possible clinical use of this virus to correct irradiation damage to salivary glands, AdhAQP1 was administered (at either 2 x 10(9) or 1 x 10(8) plaque-forming units/gland) intraductally to irradiated glands and to their contralateral nonirradiated glands. Radiation (single dose, 10 Gy) significantly reduced salivary flow in exposed glands. Virus administration resulted in gene transfer to irradiated and nonirradiated glands and was without untoward local (salivary) or systemic (sera chemistry, complete blood count) effects in all animals. However, the effect of AdhAQP1 administration varied and did not result in a consistent positive effect on salivary flow rates for all animals under these experimental conditions. We conclude that a single adenoviral-mediated gene transfer to primate salivary glands is well-tolerated, although its functional utility in enhancing fluid secretion from irradiated parotid glands is inconsistent.

Using salivary glands as a tissue target for gene therapeutics.

Gene transfer offers a potential way to correct local and systemic protein deficiency disorders by using genes as drugs, so called gene therapeutics. Salivary glands present an interesting target site for gene therapeutic applications. Herein, we review proofs of concept achieved for salivary glands with in vivo animal models. In that context we discuss problems (general and salivary tissue-specific) that limit immediate clinical use for this application of gene transfer. Ongoing efforts, however, suggest that salivary glands may be suitable as gene therapeutic target sites for drug delivery in the near future.

Salivary glands as a model for craniofacial applications of gene transfer.

The potential applications of gene transfer technology to all branches of medicine are increasing. It is quite likely that within the next 10-20 years surgical practice routinely will utilize gene transfer, at least adjunctively. The purpose of this review is to familiarize the oral and maxillofacial surgeon with this technology. Studies performed with salivary glands in animal models are presented as examples of proof of concept.

Pathologic study of a rabbit model for shigellosis.

A nonsurgical rabbit model of enteric Shigella infection was developed for studying the pathogenesis and immunology of shigellosis and for evaluating Shigella vaccine candidates. In this model, rabbits are made susceptible to Shigella infection by a pre-inoculation conditioning procedure consisting of a 36-h nonfeeding period, with 250 mg of tetracycline administered in 250 ml of drinking water, 75 mg of cimetidine given intravenously, and two 15-ml doses of 5% sodium bicarbonate given orally immediately before orogastric administration of the bacterial inoculum. Lastly 2 ml of tincture of opium is administered intraperitoneally. With a virulent strain, Shigella flexneri 2a, the clinical and pathologic characteristics of shigellosis in this rabbit model were studied. Twenty hours after oral inoculation of 10(10) bacteria, all six experimental rabbits developed diarrhea and were lethargic or moribund, whereas the four control rabbits inoculated with sterile broth remained healthy. Histologic examination revealed severe, diffuse, necrotizing ileitis with hemorrhage in experimental rabbits, whereas no lesions were found in the controls. Although the major site of necrosis in this rabbit model was the ileum, as opposed to the colon in humans and nonhuman primates, the histologic morphology of the lesion was the same in the various hosts. Because it is relatively inexpensive and convenient, this model should facilitate study of the pathophysiology and immunology of shigellosis, thereby speeding development of oral vaccines, which can be tested in this animal model.

Evidence that aquaporin-8 is located in the basolateral membrane of rat submandibular gland acinar cells.

It is possible that, during primary saliva formation, aquaporins (AQPs) facilitate transcellular water flow across acinar cells to the lumina of salivary glands. In the rat submandibular gland (rSMG) AQP5 is localized in the apical membranes of acinar cells. The presence of a basolateral AQP in the same cell type has not been reported. We have therefore used immunofluorescence confocal microscopy to determine the subcellular localization of a newly discovered aquaporin, AQP8, in rSMG epithelial cells. The antibodies we used were made against the amino- or carboxyl-terminus (anti-rAQP8NT and anti-rAQP8CT, respectively) of an AQP8 cloned from rat pancreas and liver (rAQP8). Two lines of evidence suggest that both antibodies are suitable for immunolocalization studies. First, results of immunofluorescence confocal microscopy studies show that both antibodies bind to the plasma membranes of 293 cells infected with an adenovirus encoding rAQP8. Second, results of immunoblots of membranes from infected cells suggest that both antibodies bind to glycosylated and non-glycosylated forms of rAQP8. When tested in frozen sections of rSMG, we could not detect the binding of anti-rAQP8NT to any membranes. In contrast, anti-rAQP8CT binds to the basolateral membranes of acinar (but not ductal) epithelia, suggesting that rAQP8 resides in the basolateral membranes of acinar cells. Lack of anti-rAQPNT binding to basolateral membranes suggests that this epitope is not available in the membranes. Our evidence for the basolateral localization of rAQP8 in acinar cells, coupled with previous findings that AQP5 is localized apically in the same cells, raises the possibility that water crosses the acinar epithelium through these channels during primary saliva formation.

DPC4 gene mutation in colitis associated neoplasia.

BACKGROUND: Colitis associated dysplasia and cancer often have deletions involving the long arm of chromosome 18q, suggesting the location of a tumour suppressor gene critical for their tumorigenesis. The DPC4 gene, which is genetically inactivated in pancreatic and other cancers, has recently been described. AIM: Because DPC4 is located at 18q21.1, the hypothesis that it could be a mutated tumour suppressor gene in colitis associated neoplasms was tested. PATIENTS: Advanced neoplastic lesions from six patients having chronic colitis were analysed for DPC4. METHODS: Individual exons of DPC4 were amplified by the polymerase chain reaction (PCR) and sequenced from genomic DNA of tissue specimens dissected by cryostat. RESULTS: DPC4 was found to have biallelic inactivation in one of three neoplasms shown to have allelic loss of 18q. The mutation had been acquired somatically in a plaque of high grade dysplasia. The mutation created a non-sense codon, which would cause premature termination of protein translation. CONCLUSION: The DPC4 gene is a target of 18q LOH events in colitis associated neoplasia.

Use of triplex-forming oligonucleotides and adenoviral constructs for studying the regulation of gene expression.

Short synthetic homopyrimidine- or homopurine-rich oligonucleotides can form sequence-specific triplexes with corresponding homopurine-homopyrimidine sites on duplex DNA and block transcription of a target gene in vitro. Such triplex-forming oligonucleotides (TFOs) can be rationally designed to target homopurine/homopyrimidine sequences that are often found in eukaryotic genes and thus used to modulate the expression of these genes. The antigene strategy using TFOs has been successfully applied to a number of genes in vitro. In this article we describe methods used in applying this antigene approach to the rat aquaporin 5 (rAQP5) gene. We specifically focus on the selection of TFOs based on the sequence of the target gene and on a novel method employing adenoviruses for delivery of TFOs to cells in vitro.

Evaluation of conventional media for detection of colonization factor antigens of enterotoxigenic Escherichia coli.

Strains of enterotoxigenic Escherichia coli producing either colonization factor antigen (CFA) I or II were tested for expression of CFA when grown on 16 different agar media by using agglutination and coagglutination with monoclonal antibodies, mannose-resistant hemagglutination, and a salt aggregation assay. CFA was detected from the CFA-positive strains when CFA agar was used, and it was also detected when other commercially available media were used, notably nutrient agar. CFA was not detected when other commercial media such as MacConkey agar were used. The use of nutrient agar with monoclonal antibody-based coagglutination reagents offers a potentially simple and rapid method for detecting E. coli which express CFA I or II.