Intracellular calcium: molecules and pools.

The complex nature of intracellular calcium storage pools has been examined at many levels in the past year. Additional molecules associated with calcium stores have been identified and their localization examined. The convergence of molecular biology, cell biology and biochemistry has now allowed the details of calcium signalling to be meaningfully explored.

Staged in vitro reconstitution and implantation of engineered rat kidney tissue.

A major hurdle for current xenogenic-based and other approaches aimed at engineering kidney tissues is reproducing the complex three-dimensional structure of the kidney. Here, a stepwise, in vitro method of engineering rat kidney-like tissue capable of being implanted is described. Based on the fact that the stages of kidney development are separable into in vitro modules, an approach was devised that sequentially induces an epithelial tubule (the Wolffian duct) to undergo in vitro budding, followed by branching of a single isolated bud and its recombination with metanephric mesenchyme. Implantation of the recombined tissue results in apparent early vascularization. Thus, in principle, an unbranched epithelial tubular structure (potentially constructed from cultured cells) can be induced to form kidney tissue such that this in vitro engineered tissue is capable of being implanted in host rats and developing glomeruli with evidence of early vascularization. Optimization studies (of growth factor and matrix) indicate multiple suitable combinations and suggest both a most robust and a minimal system. A whole-genome microarray analysis suggested that recombined tissue recapitulated gene expression changes that occur in vivo during later stages of kidney development, and a functional assay demonstrated that the recombined tissue was capable of transport characteristic of the differentiating nephron. The approach includes several points where tissue can be propagated. The data also show how functional, 3D kidney tissue can assemble by means of interactions of independent modules separable in vitro, potentially facilitating systems-level analyses of kidney development.

Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas.

Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with "Stains-All." Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER.

Scatter factor and the c-met receptor: a paradigm for mesenchymal/epithelial interaction.

Epithelia and mesenchyme interact during various physiologic and pathologic processes. Scatter factor is a mesenchyme-derived cytokine that stimulates motility, proliferation, and morphogenesis of epithelia. Recent studies suggest that scatter factor and its receptor (c-met) mediate mesenchyme/epithelia signalling and even interconversion. In this mini-review, we will discuss how scatter factor and c-met may mediate interactions between mesenchyme and epithelia during embryogenesis, organ repair, and neoplasia.

Molecular cloning and functional analysis of mouse C-terminal kinesin motor KifC3.

Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. To study the molecular mechanism of intracellular transport involving microtubule-dependent motors, a cDNA encoding a new kinesin-like protein called KifC3 was cloned from a mouse brain cDNA library. Sequence and secondary structure analysis revealed that KifC3 is a member of the C-terminal motor family. In contrast to other mouse C-terminal motors, KifC3 is apparently ubiquitous and may have a general role in intracellular transport. To understand the in vivo function of the KifC3 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC3 gene. Homozygous mutants of the KifC3 gene are viable, reproduce normally, and apparently develop normally. These results suggest that KifC3 is dispensable for normal development and reproduction in the mouse.

Selective amplification of protein-coding regions of large sets of genes using statistically designed primer sets.

We describe a novel approach to design a set of primers selective for large groups of genes. This method is based on the distribution frequency of all nucleotide combinations (octa- to decanucleotides), and the combined ability of primer pairs, based on these oligonucleotides, to detect genes. By analyzing 1000 human mRNAs, we found that a surprisingly small subset of octanucleotides is shared by a high proportion of human protein-coding region sense strands. By computer simulation of polymerase chain reactions, a set based on only 30 primers was able to detect approximately 75% of known (and presumably unknown) human protein-coding regions. To validate the method and provide experimental support for the feasibility of the more ambitious goal of targeting human protein-coding regions, we sought to apply the technique to a large protein family: G-protein coupled receptors (GPCRs). Our results indicate that there is sufficient low level homology among human coding regions to allow design of a limited set of primer pairs that can selectively target coding regions in general, as well as genomic subsets (e.g., GPCRs). The approach should be generally applicable to human coding regions, and thus provide an efficient method for analyzing much of the transcriptionally active human genome.

Multiple fibroblast growth factors support growth of the ureteric bud but have different effects on branching morphogenesis.

Together with glial-derived neurotrophic factor (GDNF), soluble factors present in a metanephric mesenchyme (MM) cell conditioned medium (BSN-CM) are necessary to induce branching morphogenesis of the isolated ureteric bud (UB) in vitro (Proc. Natl. Acad. Sci. USA 96 (1999) 7330). Several lines of evidence are presented here in support of a modulating role for fibroblast growth factors (FGFs) in this process. RT-PCR revealed the expression of two FGF receptors, FGFR1(IIIc) and FGFR2(IIIb), in isolated embryonic day 13 rat UBs, which by indirect immunofluorescence displayed a uniform distribution. Rat kidney organ culture experiments in the presence of a soluble FGFR2(IIIb) chimera or a neutralizing antibody to FGF7 suggested an important contribution of FGFs other than FGF7 to the branching program. Several FGFs, including FGF1, FGF2, FGF7 and FGF10, in combination with GDNF and BSN-CM were found to affect growth and branching of the isolated UB, albeit with very different effects. FGF1 and FGF7 were at extreme ends of the spectrum, with FGF10 (more FGF1-like) and FGF2 (more FGF7-like) falling in between. FGF1 induced the formation of elongated UB branching stalks with distinct proliferative ampullary tips, whereas FGF7 induced amorphous buds displaying nonselective proliferation with little distinction between stalks and ampullae. Electron microscopic examination demonstrated that FGF1 treatment induced cytoskeletal organization, intercellular junctions and lumens along the stalk portion of the developing tubules, while the ampullary regions contained 'less differentiated' cells with an abundant secretory apparatus. In contrast, FGF7-induced UBs displayed this 'less differentiated' morphology regardless of position on the structure and were virtually indistinguishable from FGF1-induced ampullae. Consistent with this, GeneChip array analysis (employing a novel nanogram-scale assay consisting of two rounds of amplification and in vitro transcription for analyzing small quantities of RNA) revealed that FGF7-induced UBs expressed more markers of cell proliferation than FGF1, which caused the UB to express cytoskeletal proteins, extracellular matrix proteins, and at least one integrin, some of which may be important in UB branch elongation. Thus, while the various FGFs examined all support UB growth, FGF1 and FGF10 appear to be more important for branching and branch elongation, and may thus play a role in determination of nephron number and patterning in the developing kidney. These in vitro data may help to explain results from knockout and transgenic studies and suggest how different FGFs may, together with GDNF and other factor(s) secreted by MM cells, regulate branching morphogenesis of the UB by their relative effects on its growth, branching and branch elongation and differentiation, thereby affecting patterning in the developing kidney.

Human Ontogeny of Drug Transporters: Review and Recommendations of the Pediatric Transporter Working Group.

The critical importance of membrane-bound transporters in pharmacotherapy is widely recognized, but little is known about drug transporter activity in children. In this white paper, the Pediatric Transporter Working Group presents a systematic review of the ontogeny of clinically relevant membrane transporters (e.g., SLC, ABC superfamilies) in intestine, liver, and kidney. Different developmental patterns for individual transporters emerge, but much remains unknown. Recommendations to increase our understanding of membrane transporters in pediatric pharmacotherapy are presented.

Development of the tubular nephron.

The renal tubule derives from two embryological structures: the metanephric mesenchyme and the ureteric bud. Tubulogenesis occurs in these two structures through somewhat different processes. The proximal through distal tubule of the nephron arises through compaction of previously unpolarized cells derived from the metanephric mesenchyme, whereas the collecting system arises through branching morphogenesis of an existing epithelial structure (the ureteric bud). Recent evidence from in vitro models using renal epithelial cells that undergo tubulogenesis and branching morphogenesis in three-dimensional collagen gels have shed light on the likely roles of growth factors, the extracellular matrix, and matrix-degrading proteinases in renal development. Differential effects of several growth factors (hepatocyte growth factor [HGF], transforming growth factor-alpha and -beta [TGF-alpha, TGF-beta], and epidermal growth factor [EGF]) suggest a mechanism for regulating the degree of tubule formation and branching events during collecting system development. Another model, the MDCK cell "calcium switch," is useful for studying the assembly of intercellular junctions and development of apical-basolateral polarity such as occurs during compaction of mesenchymally derived cells in developing renal tubules. Recent work with this model suggests that the assembly of intercellular junctions is regulated by classical signaling mechanisms including those involving intracellular calcium and calcium-dependent protein kinases. Together with organ culture studies of the embryonic kidney and analysis of genetically engineered mice, these models should allow dissection of specific molecular pathways in tubulogenesis.

Experimental studies on insecticides commonly used in India.

Using hexachlorocyclohexane (BHC) as a model histopathological, histoenzymological, biochemical, and electrophoretic studies were undertaken to find out certain parameters for early diagnosis of liver cancer. In addition, cytogenetic studies were carried out to evaluate the effect of BHC feeding on mitotic and meiotic divisions. The results of these investigations suggest that there is a significant change in liver weight in experimental group. Histologically, liver cells follow a definite sequential cellular alteration ultimately leading to liver tumor. Histochemically, well defined pattern of glycogen accumulation and iron distribution in hepatocytes was observed. The electron-microscopic observation demonstrated prominently the proliferation of agranular endoplasmic reticulum in early stages. The distribution of certain enzymes linked with plasma membrane, lysosomes, and mitochondria showed the functional alteration of these organelles both in neoplastic nodules and tumours induced by BHC. The biochemical changes observed in gluconeogenic enzymes (G6Pase and F1,6dipase) and dehydrogenases (LDH, ICDH, and MDH) at different duration of exposure to BHC indicated decrease in enzyme activity of both gluconeogenic pathway and tricarboxylic acid cycle, linked with energy metabolism. These changes tend to recover with discontinuation of BHC but 8 months continuous feeding produces irreversible changes in G6Pase activity. Using polyacrylamide gel electrophoresis technique a change in serum proteins and LDH isoenzymes was observed. However, extrapolation of these findings to human situation needs more extensive studies, taking into account all possible variables, such as the DDT and BHC load in our environment and the body burden resulting there from.

Saponins from three species of Mimusops.

Six saponins were isolated from the seed kernel of Mimusops elengi, M. hexandra and M. manilkara. Their structures were determined using a combination of 1H NMR, 13C NMR and mass spectroscopy. Three of them are new compounds: 3-O-(beta-D-glucuronopyranosyl) 28-O-(alpha-L-rhamnopyranosyl (1-->3) beta-D-xylopyranosyl(1-->4) [alpha-L-rhamnopyranosyl(1-->3)] alpha-L-rhamnopyranosyl(1-->2) alpha-L-arabinopyranosyl) protobassic acid, 3-O-(beta-D-glucuronopyranosly) 28-O-(alpha-L-rhamnopyranosyl(1-->3) beta-D-xylopyranosyl(1-->4) alpha-L-rhamnopyranosyl(1-->2) alpha-L-arabinopyranosyl) 16-alpha-hydroxyprotobassic acid and 3-O-(beta-D-glucopyranosyl(1-->3) beta-D-glucopyranosyl) 28-O-(alpha-L-rhamnopyranosyl(1-->3) beta-D-xylopyranosyl(1-->4) alpha-L-rhamnopyranosyl(1-->2) alpha-L-arabinopyranosyl) protobassic acid.

Pithedulosides A-G, oleanane glycosides from Pithecellobium dulce.

Seven saponins named pithedulosides A-G were isolated from the seeds of Pithecellobium dulce. Their structures were established through spectral analyses as echinocystic acid 3-O-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside, echinocystic acid and oleanolic acid 3-O-alpha-L-arabinopyranosyl- (1-->2)-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranosides and 3-O-beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl -(1-->6)-beta-D-glucopyranosides, oleanolic acid 3-O-alpha-L-arabinopyranosy-(1-->2)-alpha-L- arabinopyranosyl-(1-->6)-[beta-D-glucopyranosyl-(1-->2)]-beta- D-glucopyranoside, and 3-O-beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6) -[beta-D-glucopyranosyl-(1-->2)]--beta-D-glucopyranoside.

Triterpenoidal saponins from Madhuca butyracea.

Two new triterpenoidal saponins, butyrosides A and B, were isolated from the seeds of Madhuca butyracea, along with two known saponins, Mi-saponin A and 16 alpha-hydroxy Mi-saponin A. On the basis of chemical and spectroscopic evidence, the structures of butyrosides A and B were established to be 3-O-beta-D-glucopyranosyl protobassic acid 28-O-beta-D-apiofuranosyl(1----3)-beta-D-xylopyranosyl (----4)-alpha-L-rhamnopyranosyl(1----2)-alpha-L-arabinopyranoside and 3-O-beta-D-glucopyranosyl 16 alpha-hydroxy protobassic acid 28-O-beta-D-apiofuranosyl(1----3)-beta-D-xylopyranosyl (1----4)-alpha-L-rhamnopyranosyl(1----2)-alpha-L-arabinopyranoside , respectively.

Triterpenoid saponins from Madhuca butyracea.

Further investigation of the seeds of Madhuca butyracea yielded two new triterpenoid saponins, namely butyrosides C and D whose structures were established by means of chemical and spectral analyses as 3-O-beta-D-glucuronopyranosyl protobassic acid 28-O-alpha-L-rhamnopyranosyl(1-->3)-beta-D- xylopyranosyl(1-->4)-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopy ranoside and 3-O-beta-D-glucuronopyranosyl 16 alpha-hydroxy protobassic acid 28-O-beta-D-apiofuranosyl(1-->3)-beta-D-xylopyranosyl(1-->4)-alpha -L- rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside, respectively.

Early changes in serum protein and liver LDH isoenzymes in mice exposed to technical grade hexachlorocyclohexane (BHC) and their possible relationship to liver tumours.

Mice were exposed to hexachlorocyclohexane (BHC) in order to study the changes in the serum protein pattern and in the LDH isoenzymes of the liver. After 2 months of exposure the protein pattern showed a new band which persisted even after the development of a tumour. The LDH isoenzymes pattern showed a gradual decrease of the faster moving LDH-1 and LDH-2 bands which later disappeared completely when hepatic tumours formed. The significance of these results is discussed.

Effect of sensitized macrophages in transplantable sarcoma in mice. synergistic effect of hyperimmunized serum, sensitized spleen cells and macrophages in tumor transplantation.

BACKGROUND: Immunosuppression cannot develop tumors by itself. It may induce tumors under appropriate conditions and may accelerate tumor development which may be reversed by sensitized spleen cells. This study concerns the effect of sensitized macrophages in murine-transplantable sarcomas by a combination of hyperimmune serum, sensitized spleen cells and macrophages. METHODS: The main technique adopted was intraperitoneal (ip) injection of 2 mL of 10% protease peptone broth, followed 2 days later by inoculation into the peritoneum with 10-mL Hank isotonic solution. The cells from the pooled peritoneal fluid were tested by dye exclusion test to ascertain the percentage of live cells. They were tried in whole body-irradiated mice with a combination of immune serum and sensitized spleen cells to ascertain whether a suppression of growth of solid tumors could be achieved when subcutaneously (sc) administered with the previously mentioned combinations. RESULTS: The addition of immunomacrophages from transplanted tumor-bearing mice significantly suppressed the growth of subcutaneous solid tumors when the number of tumor cells was kept constant. A change in number of immunomacrophages from hyperimmunized mice at a ratio of 4:l showed a direct relationship in suppression of tumor growth. Experiments were initiated in which tumor cells were injected sc and peritoneal macrophages were injected either intravenously (iv) or ip. Experiments were then initiated to prove that cell-to-cell contact is essential for tumor suppression. In experiments in which tumor cells were administered sc and macrophages injected either iv or ip, a significant immunosuppressive effect was not shown, thus also indicating that regardless of which, cell-to-cell contact is an absolutely essential factor involved in tumor suppression. A combination of hyperimmune serum and macrophages was found to act synergistically. Macrophages and hyperimmune serum at a lesser proportion did not suppress tumor growth. Sensitized macrophages and spleen cells together significantly suppressed tumor growth in a pure isogenic strain of irradiated mice. The sensitized macrophages injected iv prolonged the survival period and retarded tumor growth. CONCLUSIONS: Tumor suppression by macrophages was found to be due to its contact with tumor cells that enables the effective transfer of immunity. Hyperimmune serum and other cells (macrophages, spleen lymphocytes) act synergistically toward each other and prolong the survival period.

Role of circulating immune complexes in the immunopathogenesis of silicosis.

The sera of 19 silica-dust-exposed subjects and of an equal number of age-, sex- and socioeconomic-strata-matched controls were analysed for antinuclear factor, rheumatoid factor, C-reactive protein, immunoglobulins G, M, A, and complement C3 and C4. Circulating immune complexes were also precipitated in all subjects and their immunoglobulin and complement C3 and C4 were estimated. Silica-exposed subjects were divided into two groups depending upon the radiological findings and it is suggested that IgA plays an important role in the immunopathogenesis of the disease and that lung changes could be due to the immune-complex-mediated mechanisms utilizing an alternative complement pathway.

Effect of immunosuppression and splenectomy in transplantation sarcomas in mice.

After spontaneous regression of transplanted tumours, marked reduction in number of tumours was found when challenged with isogenic tumour cells. The ALS abrogates this effect. Tumour removal by surgical excision of limb and subsequent time scheduled challenge by tumour cells maximally suppress on the 10th day and continues up to the 42nd day the tumorogenic effect. Splenectomy has no effect if done before a day or 3 days after challenge but marked decrease in tumour development was seen when challenged on the 8th day after splenectomy. Amputation and splenectomy together potentiates tumour formation. Only in tumour extrication, does resistance develop up to the 42nd day from surgery. Challenging at a different site in mice with tumours, resulted in prolongation of the intervals of tumour formation. Challenge after surgical removal of tumour after a time lapse, results in marked reduction in number and size of tumours. Surgical tumour extrication after splenectomy and subsequent challenge on 11th day inhibited tumour formation. Whereas splenectomized tumour bearing mice when challenged at a heterosite did not develop resistance.

Dominant-lethal study of technical-grade hexachlorocyclohexane in Swiss mice.

Male Swiss mice, 6-8 weeks old, were given a diet containing technical-grade hexachlorocyclohexane (BHC) at 500 ppm continuously for 4, 6 and 8 months. After the completion of the scheduled exposure period, the males were sequentially mated with 2-3 untreated virgin females at weekly intervals for 8 weeks. The females were autopsied at mid-term pregnancy for evaluation of dominant-lethal mutation. The number of dead implants, including deciduomas and dead embryos, showed a significant increase. Similarly, the percentage fertility and live embryos per female showed a decline when compared with the control

Humoral immunologic dysfunction in silicosis.

Immunoglobulins may play an important role in the evolution of silicosis, and their determination may serve as a helpful criterion in the diagnosis of silicosis. Serum immunoglobulin levels were studied in slate pencil workers (130) exposed to high concentrations of silica dusts and non-exposed controls (50). Significantly higher levels of immunoglobulins were observed in the silica exposed individuals. A rising trend in the serum IgG from a mean of 1373 mg/dl in control group to 2193.68 mg/dl in exposed group (conglomerate) and IgM from 140.51 mg/dl in control to 201.19 mg/dl in exposed group (conglomerate silicosis) was observed with increase in the duration of dust exposure. Highest mean levels of IgG (2193.60 mg/dl) and IgM (201.19 mg/dl) were observed in the workers having conglomerate silicosis. The results indicate that though, the levels of immunoglobulins were raised in subjects exposed to silica, this parameter may be of limited value for determining progressive of silicosis.