An anti-MUC1 antibody-calicheamicin conjugate for treatment of solid tumors. Choice of linker and overcoming drug resistance

The anti-MUC1 antibody, CTM01, has been chosen to target the potently cytotoxic calicheamicin antitumor antibiotics to solid tumors of epithelial origin that express this antigen. Earlier calicheamicin conjugates relied on the attachment of a hydrazide derivative to the oxidized carbohydrates that occur naturally on antibodies. This produced a "carbohydrate conjugate" capable of releasing active drug by hydrolysis in the lysosomes where the pH is low. Conjugates have now been made that are formed by reacting a calicheamicin derivative containing an activated ester with the lysines of antibodies. This gives an "amide conjugate" that is stable to hydrolysis, leaving the disulfide that is present in all calicheamicin conjugates as the only likely site of drug release from the conjugate. As previously shown for the carbohydrate conjugate, this amide conjugate of CTM01 produces complete regressions of xenograft tumors at doses of 300 microg/kg (calicheamicin equivalents) given three times. This indicates that hydrolytic drug release is not necessary for potent, selective cytotoxicity for calicheamicin conjugates of CTM01. Although the unconjugated calicheamicins are in general less active in cells expressing the multidrug resistance phenotype, both in vitro and in vivo results of studies reported here suggest that the efficacy of the calicheamicins toward such tumors is unexpectedly enhanced by antibody conjugation, especially for the "amide conjugate". These hydrolytically stable conjugates are also active toward cisplatin-resistant ovarian carcinoma cells as well. Such studies indicate that the calicheamicin amide conjugate of CTM01 may have potential for the treatment of MUC1-positive solid tumors, including some types of resistant tumors.     

Early alterations of brain cellular energy homeostasis in Huntington disease models

Brain energy deficit has been a suggested cause of Huntington disease (HD), but ATP depletion has not reliably been shown in preclinical models, possibly because of the immediate post-mortem changes in cellular energy metabolism. To examine a potential role of a low energy state in HD, we measured, for the first time in a neurodegenerative model, brain levels of high energy phosphates using microwave fixation, which instantaneously inactivates brain enzymatic activities and preserves in vivo levels of analytes. We studied HD transgenic R6/2 mice at ages 4, 8, and 12 weeks. We found significantly increased creatine and phosphocreatine, present as early as 4 weeks for phosphocreatine, preceding motor system deficits and decreased ATP levels in striatum, hippocampus, and frontal cortex of R6/2 mice. ATP and phosphocreatine concentrations were inversely correlated with the number of CAG repeats. Conversely, in mice injected with 3-nitroproprionic acid, an acute model of brain energy deficit, both ATP and phosphocreatine were significantly reduced. Increased creatine and phosphocreatine in R6/2 mice was associated with decreased guanidinoacetate N-methyltransferase and creatine kinase, both at the protein and RNA levels, and increased phosphorylated AMP-dependent protein kinase (pAMPK) over AMPK ratio. In addition, in 4-month-old knock-in Hdh(Q111/+) mice, the earliest metabolic alterations consisted of increased phosphocreatine in the frontal cortex and increased the pAMPK/AMPK ratio. Altogether, this study provides the first direct evidence of chronic alteration in homeostasis of high energy phosphates in HD models in the earliest stages of the disease, indicating possible reduced utilization of the brain phosphocreatine pool.     

Identification of the SPG15 gene, encoding spastizin, as a frequent cause of complicated autosomal-recessive spastic paraplegia, including Kjellin syndrome

Hereditary spastic paraplegias (HSPs) are genetically and phenotypically heterogeneous disorders. Both "uncomplicated" and "complicated" forms have been described with various modes of inheritance. Sixteen loci for autosomal-recessive "complicated" HSP have been mapped. The SPG15 locus was first reported to account for a rare form of spastic paraplegia variably associated with mental impairment, pigmented maculopathy, dysarthria, cerebellar signs, and distal amyotrophy, sometimes designated as Kjellin syndrome. Here, we report the refinement of SPG15 to a 2.64 Mb genetic interval on chromosome 14q23.3-q24.2 and the identification of ZFYVE26, which encodes a zinc-finger protein with a FYVE domain that we named spastizin, as the cause of SPG15. Six different truncating mutations were found to segregate with the disease in eight families with a phenotype that included variable clinical features of Kjellin syndrome. ZFYVE26 mRNA was widely distributed in human tissues, as well as in rat embryos, suggesting a possible role of this gene during embryonic development. In the adult rodent brain, its expression profile closely resembled that of SPG11, another gene responsible for complicated HSP. In cultured cells, spastizin colocalized partially with markers of endoplasmic reticulum and endosomes, suggesting a role in intracellular trafficking.     

Protein kinase C and leucine metabolism in intestinal crypt cells

Intestinal cells were separated into fractions rich in villous or crypt cells. Alkaline phosphatase, present in villous cells, but absent from crypt cells, was used as a marker. Crypt cells were 3-6 times as active as villous cells in the metabolism of leucine or mevalonate. The previously reported stimulatory effect of albumin was twice as strong in crypt cells as that in villous cells. Reduced glutathione, spermidine HCl and ethanolamine (0.5-10 mM) did not replace albumin, the effect of which was maximal at 0.02 mM. Protein kinase C was shown to be present mainly in crypt cells.     

Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies

A thorough understanding of nerve regeneration in Caenorhabditis elegans requires performing femtosecond laser nanoaxotomy while minimally affecting the worm. We present a microfluidic device that fulfills such criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using the 'nanoaxotomy' chip, we discovered that axonal regeneration occurs much faster than previously described, and notably, the distal fragment of the severed axon regrows in the absence of anesthetics.