Haemopoietic growth factors: their role in cell development and their clinical use

Mature blood cells are derived from haemopoietic stem cells which grow and proliferate to give rise to progenitor cells more restricted in their proliferation and differentiation capacity. These in turn give rise to cells belonging to any of the haemopoietic lineages. The haemopoietic growth factors interleukin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony stimulating factor, macrophage colony-stimulating factor and erythropoietin act on haemopoietic cells to promote cell survival, proliferation, differentiation and maturation, as well as many functions of the mature cells. These factors, now purified to homogeneity and molecularly cloned have recently become available. This has facilitated studies of their roles in cell production, and the range of target cells sensitive to them in vitro and in vivo in several species. The latter experimental data led to the first clinical trials where these factors have been used successfully in several clinical settings: erythropoietin to correct the anaemia of renal disease; granulocyte and granulocyte-macrophage colony-stimulating factors to accelerate haemopoietic regeneration after chemotherapy and bone marrow transplantation, and in other situations where increase in the numbers of white cells and stimulation of their function were required. The results to date allow optimism; the clinical use of growth factors not only in haematology and oncology, but in wider fields of medicine may well constitute a major breakthrough in the near future.     

The benthic invertebrates of the Salton Sea: distribution and seasonal dynamics

The Salton Sea, California's largest inland water body, is an athalassic saline lake with an invertebrate fauna dominated by marine species. The distribution and seasonal dynamics of the benthic macroinvertebrate populations of the Salton Sea were investigated during 1999 in the first survey of the benthos since 1956. Invertebrates were sampled from sediments at depths of 2–12 m, shallow water rocky substrates, and littoral barnacle shell substrates. The macroinvertebrates of the Salton Sea consist of a few invasive, euryhaline species, several of which thrive on different substrates. The principal infaunal organisms are the polychaetes Neanthes succinea Frey & Leuckart and Streblospio benedicti Webster, and the oligochaetes Thalassodrilides gurwitschi Cook, T. belli Hrabe, and an enchytraeid. All but Neanthes are new records for the Sea. Benthic crustacean species are the amphipods Gammarus mucronatus Say, Corophium louisianum Shoemaker, and the barnacle Balanus amphitrite Darwin. Neanthes succinea is the dominant infaunal species on the Sea bottom at depths of 2–12 m. Area-weighted estimates of N. succinea standing stock in September and November 1999 were two orders of magnitude lower than biomass estimated in the same months in 1956. During 1999, population density varied spatially and temporally. Abundance declined greatly in offshore sediments at depths >2 m during spring and summer due to decreasing oxygen levels at the sediment surface, eventually resulting in the absence of Neanthes from all offshore sites >2 m between July and November. In contrast, on shoreline rocky substrate, Neanthes persisted year round, and biomass density increased nearly one order of magnitude between January and November. The rocky shoreline had the highest numbers of invertebrates per unit area, exceeding those reported by other published sources for Neanthes, Gammarus mucronatus, Corophium louisianum, and Balanus amphitrite in marine coastal habitats. The rocky shoreline habitat is highly productive, and is an important refuge during periods of seasonal anoxia for Neanthes and for the other invertebrates that also serve as prey for fish and birds.     

Investigation of aryl isonitrile compounds with potent,broad-spectrum antifungal activity

Invasive fungal infections present a formidable global public health challenge due to the limited number of approved antifungal agents and the emergence of resistance to the frontline treatment options, such as fluconazole. Three fungal pathogens of significant concern are Candida, Cryptococcus, and Aspergillus given their propensity to cause opportunistic infections in immunocompromised individuals. New antifungal agents composed of unique chemical scaffolds are needed to address this public health challenge. The present study examines the structure-activity relationship of a set of aryl isonitrile compounds that possess broad-spectrum antifungal activity primarily against species of Candida and Cryptococcus. The most potent derivatives are capable of inhibiting growth of these key pathogens at concentrations as low as 0.5 µM. Remarkably, the most active compounds exhibit an excellent safety profile and are non-toxic to mammalian cells even at concentrations up to 256 µM. The present study lays the foundation for further investigation of aryl isonitrile compounds as a new class of antifungal agents.     

DNA sequence recognition in the minor groove by hairpin pyrrole polyamide-Hoechst 33258 analogue conjugate

A hairpin pyrrole polyamide conjugated to a Hoechst 33258 (Ht) analogue, PyPyPy-gamma-PyPyPy-gamma-Ht, was synthesized on solid-phase by adaptation of an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. Sequence selectivity and complex stabilities were characterized by spectrofluorometric titrations and thermal melting studies. The polyamide of the conjugate was observed to bind in a hairpin motif forming 1:1 conjugate:dsDNA complexes. The conjugate is able to recognize nine contiguous A/T bps, discriminating from the sequences containing fewer than nine contiguous A/T bps.     

Pressure stability of proteins at their isoelectric points

Yeast alcohol dehydrogenase is slowly denatured at moderate hydrostatic pressure (t(1/2) approximately equals 30 min at 2 kbar and pH 7). The extent of denaturation is pH dependent with maximal stability near the isoelectric point of the protein, pH = 5.4. While not a surprising finding, it appears that this phenomenon has not been documented before (or at least not identified) despite many investigations into the pressure stability of proteins. Consideration of changes in the net charge of proteins far from their isoelectric points may explain other pressure effects as well.     

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells

Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.     

Molecular, biochemical and functional analysis of a novel and developmentally important fibrillar collagen (Hcol-I) in hydra

The body wall of hydra (a member of the phylum Cnidaria) is structurally reduced to an epithelial bilayer with an intervening extracellular matrix (ECM). Previous studies have established that cell-ECM interactions are important for morphogenesis and cell differentiation in this simple metazoan. The ECM of hydra is particularly interesting because it represents a primordial form of matrix. Despite progress in our understanding of hydra ECM, we still know little about the nature of hydra collagens. In the current study we provide a molecular, biochemical and functional analysis of a hydra fibrillar collagen that has similarity to vertebrate type I and type II collagens. This fibrillar collagen has been named hydra collagen-I (Hcol-I) because of its structure and because it is the first ECM collagen to be identified in hydra. It represents a novel member of the collagen family. Similar to vertebrate type I and II collagens, Hcol-I contains an N-terminal propeptide-like domain, a triple helical domain containing typical Gly-X-Y repeats and a C-terminal propeptide domain. The overall identity to vertebrate fibrillar collagens is about 30%, while the identity of the C-terminal propeptide domain is 50%. Because the N-terminal propeptide domain is retained after post-translational processing, Hcol-I does not form thick fibers as seen in vertebrates. This was confirmed using transmission electron microscopy to study rotary shadow images of purified Hcol-I. In addition, absence of crucial lysine residues and an overall reduction in proline content, results in reduced crosslinking of fibrils and increased flexibility of the molecule, respectively. These structural changes in Hcol-I help to explain the flexible properties of hydra ECM. Immunocytochemical studies indicate that Hcol-I forms the 10 nm fibrils that comprise the majority of molecules in the central fibrous zone of hydra ECM. The central fibrous zone resides between the two subepithelial zones where hydra laminin is localized. While previous studies have shown that basal lamina components like laminin are expressed by the endoderm, in situ hybridisation studies show that Hcol-I mRNA expression is restricted to the ectoderm. Hcol-I expression is upregulated during head regeneration, and antisense studies using thio-oligonucleotides demonstrated that blocking the translation of Hcol-I leads to a reversible inhibition of head morphogenesis during this regenerative process. Taken in total, the data presented in this study indicate that Hcol-I is required for morphogensis in hydra and represents a novel fibrillar collagen whose structural characteristics help to explain the unique biophysical properties of hydra ECM. Interestingly, the structure of Hcol-I mimics what is seen in Ehlers-Danlos syndrome type VII in humans; an inherited pathological condition that leads to joint and skin abnormalities. Hcol-I therefore illustrates an adaptive trait in which the normal physiological situation in hydra translates into a pathological condition in humans.     

Non-invasive genotyping of Helicobacter pylori cagA, vacA, and hopQ from asymptomatic children

Background: Helicobacter pylori infection is usually acquired in childhood, but little is known about its natural history in asymptomatic children, primarily due to the paucity of non‐invasive diagnostic methods. H. pylori strains harboring cagA and specific alleles of hopQ and vacA are associated with increased risk for gastric cancer. Many studies of H. pylori virulence markers in children have the bias that symptomatic subjects are selected for endoscopy, and these children may harbor the most virulent strains. Our aim is to genotype cagA, hopQ, and vacA alleles in stool DNA samples of healthy Colombian children residing in an area with high incidence of gastric cancer, to avoid selection bias resulting from endoscopy. Methods: H. pylori status of 86 asymptomatic children was assessed by ¹³C‐urea breath test (UBT) and PCR. H. pylori 16S rRNA, cagA, hopQ, and vacA genes were amplified from stool DNA samples and sequenced. Results: UBT was positive in 69 (80.2%) of 86 children; in stool DNA analysis, 78.3% were positive by 16S rRNA PCR. cagA, vacA, and hopQ were detected in 66.1%, 84.6%, and 72.3% of stool DNA samples from 16S rRNA‐positive children. Of the children’s DNA samples, which revealed vacA and hopQ alleles, 91.7% showed vacA s1 and 73.7% showed type I hopQ. Type I hopQ alleles were associated with cagA positivity and vacA s1 genotypes (p < 0.0001). Conclusions: Using stool DNA samples, virulence markers of H. pylori were successfully genotyped in a high percentage of the asymptomatic infected children, revealing a high prevalence of genotypes associated with virulence. Type I hopQ alleles were associated with the presence of cagA and the vacA s1 genotype.