Biodistribution of 111In-labelled engineered human antibody CTM01 (hCTM01) in ovarian cancer patients: influence of prior administration of unlabelled hCTM01

mAb hCTM01 binds a carcinoma-associated antigen, the MUC1 gene product. The antigen is also present in the circulation, and administration of ¹¹¹In-labelled hCTM01 results in the formation of immune complexes with enhanced accumulation in the liver. To avoid the unwanted effect of circulating radioactive immune complexes, a strategy to remove the circulating antigen was investigated using a split-dosage schedule. Eleven patients suspected of having ovarian carcinoma were injected with 1 mg/kg unlabelled hCTM01, 1 h before receiving 0.1 mg/kg ¹¹¹In-labelled hCTM01 (100 MBq). The amount of radioactivity was determined in resected tumour tissue, various normal tissues and blood samples obtained at laparotomy 6 days postinjection (p.i.). In all patients, the circulating antigen decreased to its nadir after the unlabelled antibody infusion and immune complex formation was demonstrated. Uptake in tumour deposits 6 days p.i. was 11.1 times higher than in normal tissues (P<0.0001) and 5.9 times higher than in blood (P<0.0001). ¹¹¹In activity in liver tissue was comparable to ¹¹¹In uptake in tumour tissue, and considerably lower than previously reported in patients not pretreated with unlabelled antibody. The split-dosing strategy would appear to be advantageous for use of hCTM01 as a specific carrier for the delivery of cytotoxic agents to patients with ovarian cancer. Received: 12 February 1998 / Accepted: 30 April 1998     

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

EMG median power frequency in an exhausting exercise

EMG median power frequency of the calf muscles was investigated during an exhausting treadmill exercise. This exercise was an uphill run, the average endurance time was 1.5 min. Median power frequency of the calf muscles declined by more than 10% during this exercise. In addition EMG median power frequency of isometric contractions of the same muscles was measured before and in one minute intervals for 10 min after this run. Immediately after the run isometric median power frequency had declined by less than 5% for the soleus muscle, more than 10% for the gastrocnemius medialis and gastrocnemius lateralis muscles. In the 10 min following exercise the isometric median power frequency increased to pre-execise levels. Maybe the median power frequency shift to lower frequencies during dynamic exercise can be interpreted as a sign of local muscle fatigue.     

Fed-batch cultivation of Pseudomonas cepacia with on-line control of the toxic substrate salicylate

Pseudomonas cepacia was cultivated on salicylate as sole carbon and energy source in a fed-batch culture. On-line measurement of salicylate concentration was carried out using a filtration system. Cell-free permeate was passed through a flow-through spectrophotometer. Measurements were carried out at 325 nm. A Proportional-Integral controller was used for regulating the feed rate around a basic dosage scheme to maintain a constant salicylate concentration of 0.2 g/l in the broth. The cell mass concentration increased from less than 1 g dry weight (dw)/l to 12 g dw/l in less than 6 h, and the yield coefficient was 0.4 g dw/g salicylate. The intracellular enzymatic activity of salicylate hydroxylase was virtually unchanged during the fed-batch cultivation. Correspondence to: A. Tocaj     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

In the higher plant Pisum sativum maturation of nascent DNA is blocked by cycloheximide, but only after 4-8 replicons are joined.

Velocity sedimentation in alkaline sucrose gradients, single cell autoradiography and cytophotometry were used to determine if protein synthesis is required for the maturation of nascent replicons to chromosomal-sized molecules in cultured pea-root cells. The results obtained showed that cycloheximide at 5 and 10 microgram/ml, added either before or during labeling with tritiated thymidine, blocked maturation of nascent DNA at an intermediate size of 72-140 X 10(6) daltons single-stranded DNA. To reach this size, nascent replicons - which are 18 X 10(6) daltons single-stranded DNA each - were replicated and groups of 4-8 replicons were joined even though protein synthesis was reduced to 15% of the control. Further maturation of the nascent molecules to chromosomal size, however, was prevented and this resulted in the accumulation of nascent molecules in the 72-140 X 10(6) daltons range. The experiments also showed that the joining of nascent replicons is not an absolute function of late S or G2 phase of the cell cycle, since cells treated with cycloheximide and blocked in mid-S phase had nascent DNA of a size corresponding to 4-8 joined replicons. Finally, the results support the hypothesis that at least one step in the process of nascent DNA maturation may require replication, during late-S phase, of DNA segments that are interspersed within replicon-clusters that replicate early in the S phase.     

Brain structure variation in great apes, with attention to the mountain gorilla (Gorilla beringei beringei)

This report presents data regarding the brain structure of mountain gorillas (Gorilla beringei beringei) in comparison with other great apes. Magnetic resonance (MR) images of three mountain gorilla brains were obtained with a 3T scanner, and the volume of major neuroanatomical structures (neocortical gray matter, hippocampus, thalamus, striatum, and cerebellum) was measured. These data were included with our existing database that includes 23 chimpanzees, three western lowland gorillas, and six orangutans. We defined a multidimensional space by calculating the principal components (PCs) from the correlation matrix of brain structure fractions in the well-represented sample of chimpanzees. We then plotted data from all of the taxa in this space to examine phyletic variation in neural organization. Most of the variance in mountain gorillas, as well as other great apes, was contained within the chimpanzee range along the first two PCs, which accounted for 61.73% of the total variance. Thus, the majority of interspecific variation in brain structure observed among these ape taxa was no greater than the within-species variation seen in chimpanzees. The loadings on PCs indicated that the brain structure of great apes differs among taxa mostly in the relative sizes of the striatum, cerebellum, and hippocampus. These findings suggest possible functional differences among taxa in terms of neural adaptations for ecological and locomotor capacities. Importantly, these results fill a critical gap in current knowledge regarding great ape neuroanatomical diversity.     

The condition for dynamic stability

The well-known condition for standing stability in static situations is that the vertical projection of the centre of mass (CoM) should be within the base of support (BoS). On the basis of a simple inverted pendulum model, an extension of this rule is proposed for dynamical situations: the position of (the vertical projection of) the CoM plus its velocity times a factor (square root l/g) should be within the BoS, l being leg length and g the acceleration of gravity. It is proposed to name this vector quantity 'extrapolated centre of mass position' (XcoM). The definition suggests as a measure of stability the 'margin of stability' b, the minimum distance from XcoM to the boundaries of the BoS. An alternative measure is the temporal stability margin tau, the time in which the boundary of the BoS would be reached without intervention. Some experimental data of subjects standing on one or two feet, flatfoot and tiptoe, are presented to give an idea of the usual ranges of these margins of stability. Example data on walking are also presented.     

Exogenous melatonin reduces the resynchronization time after phase shifts of a nonphotic zeitgeber in the house sparrow (Passer domesticus)

Continuous melatonin administration via silastic implants accelerates the resynchronization of the circadian locomotor activity rhythm in house sparrows (Passer domesticus) after exposure to phase shifts of a weak light-dark cycle. Constant melatonin might induce this effect either by increasing the sensitivity of the visual system to a light zeitgeber or by reducing the degree of self-sustainment of the circadian pacemaker. To distinguish between these two possible mechanisms, two groups of house sparrows, one carrying melatonin implants and the other empty implants, were kept in constant dim light and subjected to advance and delay shifts of a 12-h feeding phase. The resynchronization times of their circadian feeding rhythm following the phase shifts were significantly shorter when the birds carried melatonin implants than when they carried empty implants. In a second experiment, melatonin-implanted and control birds were released into food ad libitum conditions 2 days after either a delay or an advance phase shift. The number of hours by which the activity rhythms had been shifted on the second day in food ad libitum conditions was assessed. Melatonin-implanted house sparrows had significantly larger phase shifts in their circadian feeding rhythm than control birds. This is in accordance with the first experiment since a larger phase shift at a given time reflects accelerated resynchronization. Additionally, the second experiment also excludes any possible masking effects of the nonphotic zeitgeber. In conclusion, constant melatonin accelerates resynchronization even after phase shifts of a nonphotic zeitgeber, indicating that constant high levels of melatonin can reduce the degree of self-sustainment of the circadian pacemaker independent of any effects on the photoreceptive system.