A novel method for human hematopoietic stem/progenitor cell isolation from umbilical cord blood based on immunoaffinity aqueous two-phase partitioning

A novel cell separation process based on immunoaffinity aqueous two phase systems is presented to isolate and purify CD34⁺ stem/progenitor cells directly from the whole umbilical cord blood (UCB). A system, composed of polyethylene glycol and dextran, was evaluated for the selective recovery of CD34⁺ cells from UCB. A monoclonal antibody against the CD34 surface antigen was used for the direct partitioning of CD34⁺ cells in UCB to the PEG-rich phase. The initial population of CD34⁺ cells (0.2% of the initial sample) was enriched to values up to 42% in a single partitioning step, while the majority of contaminant cells were partitioned to the dextran-rich phase (1.37 × 10⁻² < K_P < 2.76 × 10⁻²). This novel selection method allowed a recovery yield of 95% of CD34⁺ cells with a purification factor of 245 and is expected to pave a new way to purify hematopoietic stem/progenitor cells for use in a variety of clinical settings.     

Cell-cycle-dependent regulation of CNT1, a concentrative nucleoside transporter involved in the uptake of cell-cycle-dependent nucleoside-derived anticancer drugs

Most nucleoside-derived anticancer drugs are taken up by the high-affinity Na-dependent nucleoside transporter CNT1. Since such drugs are to some extent cell-cycle-dependent in their cytotoxic action, we examined the relationship between CNT1 expression and cell-cycle progression in the rat hepatoma cell line FAO. Cell cultures were synchronized either at late G1 or early S stages by combining mimosin treatment with either previous synchronization or not by serum starvation. Cell-cycle progression was then assessed by measuring [methyl-3H]thymidine incorporation into DNA and monitoring cyclin E and A protein levels. In these conditions, CNT1 protein amounts increase at the G1-S transition. When cells were synchronized using hydroxyurea (HU), which directly interacts with nucleotide metabolism by inhibiting ribonucleotide reductase, CNT1 protein amounts increased in synchronized cells and remained high during cell-cycle progression. These data indicate that CNT1 adapts to cell-cycle progression and responds to nucleos(t)ide metabolism status, a feature that might contribute to the cytotoxic action of cell-cycle-dependent anticancer drugs.     

Identification of Novel Imidazo[1,2-a]pyridine Inhibitors Targeting M. tuberculosis QcrB

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1–4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism ⁹³⁷ACC>⁹³⁷GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.     

Permanent contraception of dogs induced with intratesticular injection of a Zinc Gluconate-based solution

The objective was to evaluate the efficacy of a single intratesticular injection of a Zinc Gluconate-based solution to induce sterility in male dogs. Fifteen pubertal mongrel dogs (8 mo to 4 y old) were assigned to two groups; Control dogs (n = 5) received a single injection of an isotonic saline solution into each testis, whereas Treated dogs (n = 10), were given Testoblock, a proprietary zinc-gluconate (13.1 mg zinc/ml) solution in a physiological vehicle. The volume of saline or Testoblock injected varied from 0.2 to 1.0 ml/testis (based on testis width). Physical examination, testis width, hematology, clinical chemistry (hepatic and renal function), plasma testosterone concentration, semen characteristics, and libido, were assessed until castration (150 d after treatment). In Treated dogs, testis width increased (approximately 20%) relative to that in Control dogs, but subsequently was not significantly different from Controls (group × time interaction, P < 0.0001). For all dogs, values for hematology and clinical chemistry consistently remained within reference ranges. Although plasma testosterone concentrations decreased over time (P < 0.006), there was only a tendency for an effect of group (P < 0.09), and libido was not significantly affected. By 63 d after Testoblock treatment, eight Treated dogs were azoospermic, whereas the remaining two were oligozoospermic (<10 × 10⁶ sperm/ml). We concluded that intratesticular injection of the Zinc Gluconate-based chemical sterilant Testoblock has considerable potential to induce permanent contraception in male dogs.     

Immobilization and stabilization of a bimolecular aggregate of the lipase from Pseudomonas fluorescens by multipoint covalent attachment

The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 °C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol.