Fluid-phase endocytosis does not contribute to rapid fluid secretion in the malpighian tubules of the house cricket, Acheta domesticus.

When the Malpighian tubules (Mt) of the house cricket (Acheta domesticus) are treated with dibutyryl adenosine 3', 5'-cyclic monophosphate (db-cAMP; 1 mM), which causes a doubling in secretion rate, more than 50% of the cell volume is occupied by vesicles within 420 sec of exposure. In view of the fact that the increase in vesiculation occurs concomitantly with stimulated fluid transport, we set out to determine whether the vesicles are formed as a result of fluid-phase endocytosis (pinocytosis) and subsequently used to transport fluid to the lumen as one means of increasing transport rate. We used fluorescent fluid-phase markers (Lucifer Yellow Carbohydrazide [LYCH] and Alexa 488 hydrazide) and an electron dense marker (cationized ferritin) to elucidate the degree of endocytosis that occurred with db-cAMP stimulation. We found that, although some fluid is taken into the cells of the mid-tubule via endocytosis, it does not coincide with the level of vacuolation present in stimulated tubules. The amount of LYCH transported into the primary urine by the db-cAMP-stimulated Mt decreased by 40% as compared to the unstimulated transport, and the rate of transport of LYCH was only 30% of the unstimulated tubules. In summary, our findings do not support the theory that the majority of the vesicles or vacuoles comprise intracellular, endocytotic compartments formed via a basolateral endocytotic pathway. We also found no evidence to support the functioning of vesicles or vacuoles as transcellular "shuttling" mechanisms to move fluid from the basal region to the apical membrane and into the lumen.     

Enhancement of therapeutic protein in vivo activities through glycoengineering

Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).     

Retrospective analysis of a local cessation of vaccination against poliomyelitis: a possible scenario for the future

The global eradication of poliomyelitis will require substantial changes in immunization practices. One of the proposed scenarios includes cessation of vaccination with live oral poliovirus vaccine (OPV) and the creation of an OPV stockpile for emergency response in case of the reintroduction of poliovirus into circulation. We describe here a retrospective analysis of the cessation of OPV usage in a region of the Byelorussian Republic of the former Soviet Union in 1963 to 1966. During this period, a widespread circulation and evolution of independent lineages of vaccine-derived polioviruses took place in the region. Some of these lineages appeared to originate from OPV given to 40 children in the community during this period of essentially no vaccinations. The data demonstrate very high risks associated with both the local cessation of OPV vaccination and the proposed use of OPV to control a possible reemergence of poliovirus in the postvaccination period. The high transmissibility of OPV-derived viruses in nonimmune population, documented here, and the known existence of long-term OPV excretors should be also considered in assessing risks of the synchronized global cessation of OPV usage.     

A finite difference model of O2 transport in aortic valve cusps: importance of intrinsic microcirculation

Recent studies have reported the presence of a microcirculation within the tissue of aortic valves. To test the hypothesis that this vascular bed is needed to satisfy the oxygen demands of the cusp tissue, a two-dimensional (2D) finite difference model of oxygen diffusion was developed. The in vivo environment was modeled for vascular and avascular cusps using thickness data from precise radiographic measurements of fresh porcine valves, and O2 diffusivity (DO2) and O2 consumption (VO2) values from experimental data. The location and density of the cusp vasculature were determined by the model to prevent oxygen levels from falling to zero. Validation of the model was performed by simulation of the experimental measurements of cusp DO2 and VO2. For a test cusp with uniform thickness, the model returned simulated DO2 and VO2 measurements within 1.43% and 0.18% difference of the true parameter values, respectively. For native cusps, the simulated DO2 measurements were sensitive to thickness variations (-38 to +21% difference), whereas the VO2 measurements were minimally affected (8% difference). An improved DO2 measurement technique was found to reduce these errors to <5% and is recommended for analysis of experimental data. In the avascular case, the model predicted large regions of hypoxic tissue, whereas in the vascular case, the model predicted vessel locations and densities similar to what was experimentally observed in porcine cusps. Overall, the in vivo model developed in this study confirmed the need for an intrinsic microcirculation in the thicker basal regions of aortic cusps.     

Avipox sp. in a colony of gray-crowned rosy finches (Leucosticte tephrocotis)

Members of a wild-caught colony of 16 gray-crowned rosy finches (Leucosticte tephrocotis) were presented with dermal and mucosal lesions, anorexia, emaciation, lethargy, and sudden death. Lesions included dermatitis, conjunctivitis, and glossitis. Skin scrapings from and bacterial culture of dermal lesions yielded Staphylococcus aureus and Candida albicans. Necropsy and histologic examination revealed characteristic epidermal and mucosal pox lesions, with the presence of characteristic Bollinger body intracellular inclusions. Electron microscopy (EM) provided confirmation of pox virus infection. This epornitic resulted in the death or euthanasia of 12 birds (75% morbidity and associated mortality) and was brought to conclusion through culling of affected birds. The source of infection remains unknown, although multiple modes of introduction exist. Similar epornitics may be prevented through indoor, species-specific housing, and quarantine. Vaccination and antiparasitic treatment may reduce the risk of disease spread.     

Engineering S-protein fragments of bovine ribonuclease A for targeted drug delivery

High affinity interaction between S-protein and S-peptide fragments of bovine pancreatic RNase A has been recently used for construction of molecular vehicles for targeted drug delivery. The vehicle is assembled as a complex of drug carrier conjugated S-protein with S-peptide-tagged targeting protein. To avoid random chemical crosslinking of drug carriers to S-protein, we constructed a mutant 16-124aa fragment of RNase A in which 122ala is replaced with a cysteine residue. The mutant and the corresponding wild type fragments expressed in Escherichia coli are refolded into functional conformations only in the presence of S-peptide. After the removal of S-peptide, both fragments retain the ability to bind S-peptide and S-peptide-tagged proteins. The 122cys residue in the mutant fragment is available for site-specific conjugation.     

Investigations of proliferative and senescent callus of soybean

Different explant sources from several Giycine max (L.) Merr. cultivars and several G. soja F. J. Herm Plant Introductions were tested for calluas production. Embryo axis explants produced callus most readily; hence, they were selected for media response studies and microscopic evaluation. Two callus types, proliferative and senescing, were identified and characterized by light and scanning electron microscopy (SEM). Proliferative callus was composed of chloroplast-containing cells interspersed with colorless meristematic areas, while senescing callus contained mostly large, vacuolate cells without chloroplasts. In SEM preparations a furry, layered, mucilaginous coating was seen on the outer surface of the senescing callus, while proliferative callus cells had uncoated, smooth or wrinkled surfaces. The mucilage contained glucose and galactose.     

A Drosophila model of multiple endocrine neoplasia type 2

Dominant mutations in the Ret receptor tyrosine kinase lead to the familial cancer syndrome multiple endocrine neoplasia type 2 (MEN2). Mammalian tissue culture studies suggest that RetMEN2 mutations significantly alter Ret-signaling properties, but the precise mechanisms by which RetMEN2 promotes tumorigenesis remain poorly understood. To determine the signal transduction pathways required for RetMEN2 activity, we analyzed analogous mutations in the Drosophila Ret ortholog dRet. Overexpressed dRetMEN2 isoforms targeted to the developing retina led to aberrant cell proliferation, inappropriate cell fate specification, and excessive Ras pathway activation. Genetic analysis indicated that dRetMEN2 acts through the Ras-ERK, Src, and Jun kinase pathways. A genetic screen for mutations that dominantly suppress or enhance dRetMEN2 phenotypes identified new genes that are required for the phenotypic outcomes of dRetMEN2 activity. Finally, we identified human orthologs for many of these genes and examined their status in human tumors. Two of these loci showed loss of heterozygosity (LOH) within both sporadic and MEN2-associated pheochromocytomas, suggesting that they may contribute to Ret-dependent oncogenesis.     

The genomic island SGI1, containing the multiple antibiotic resistance region of Salmonella enterica serovar Typhimurium DT104 or variants of it, is widely distributed in other S. enterica serovars

The global dissemination of the multiply-antibiotic-resistant Salmonella enterica serovar Typhimurium DT104 clone with the resistance genes located in a class 1 integron, here designated In104, within genomic island SGI1 is a significant public health issue. Here, we have shown that SGI1 and variants of it carrying different combinations of resistance genes are found in several Salmonella enterica serovars. These are serovars Cerro, Derby, Dusseldorf, Infantis, Kiambu, and Paratyphi B dT(+) isolated from human infections and serovar Emek from sewage effluent. Two new variants, SGI1-I and SGI1-J, both of which include the dfrA1-orfC cassette array, were identified.     

Placental improvement and reduced distal limb defects by maternal interferon-gamma injection in methylnitrosourea-exposed mice

BACKGROUND: Methylnitrosourea (MNU), an alkylating agent derived from creatinine metabolism, is cytotoxic, genotoxic, and mutagenic. Mid-gestational exposure to MNU leads to distal limb defects in mice. Previous studies have shown that nonspecific maternal immune stimulation protects against MNU-induced teratogenesis. A role for immune-mediated placental improvement in this effect remains uncertain. METHODS: The immune system of timed-pregnant C57BL/6N and CD-1 mice was stimulated by GD 7 intraperitoneal (IP) injection with the cytokine interferon-gamma (IFN-gamma). A teratogenic dose of MNU was then administered by IP injection on the morning of GD 9 to disrupt distal limb formation. Fetal limb length, body length, digital deformities, and placental integrity were evaluated on GD 14. RESULTS: The incidence of syndactyly, polydactyly, and interdigital webbing in MNU-exposed mice was decreased by maternal IFN-gamma treatment. In C57BL/6N mice, these defects were reduced by 47, 100, and 63%, respectively, as compared to previous reports on CD-1 mice, by 39, 71, and 20%, respectively. Administration of IFN-gamma significantly diminished MNU-induced endothelial and trophoblast placental damage in both strains of mice. CONCLUSIONS: These findings support a possible link between maternal immunity, placental integrity, and fetal distal limb development. Further, these results suggest that IFN-gamma might act through placental improvement to indirectly protect against MNU-induced fetal limb malformations.