Suppression of tumorigenicity and metastasis of human renal carcinoma cells by infection with retroviral vectors harboring the murine inducible nitric oxide synthase gene

The purpose of this study was to determine whether retrovirus-mediated transfer of the murine macrophage inducible nitric oxide synthase (iNOS) gene can inhibit tumorigenicity and metastasis of human renal cancer cells. Retroviral vectors encoding murine macrophage iNOS were constructed in the pLXSN retroviral vector with the iNOS gene under the control of a long terminal repeat promoter and a neomycin resistance gene under the control of an internal simian virus 40 promoter. Highly metastatic human renal carcinoma SN12PM6 cells were infected with control or iNOS retrovirus. Expression of iNOS was confirmed by Northern and Western blot analyses, and expression of the functional iNOS protein, i.e., production of nitric oxide (NO), was determined by measuring nitrite accumulation in culture supernatants. Noninfected or control cells produced large orthotopic tumors in the kidney of nude mice and a larger number of experimental lung metastases, whereas iNOS-infected cells produced small tumors in the kidneys and few to no lung metastases. The data indicate that the infection of human renal cancer cells by retroviruses harboring the murine iNOS gene can induce the production of high levels of NO, which is associated with autocytotoxicity, suppression of tumorigenicity, and abrogation of metastasis.     

Calcium-sensitive fluorescent dyes can report increases in intracellular free zinc concentration in cultured forebrain neurons

High concentrations of Zn2+ are found in presynaptic terminals of excitatory neurons in the CNS. Zn2+ can be released during synaptic activity and modulate postsynaptic receptors, but little is known about the possibility that Zn2+ may enter postsynaptic cells and produce dynamic changes in the intracellular Zn2+ concentration ([Zn2+]i). We used fura-2 and magfura-2 to detect the consequences of Zn2+ influx in cultured neurons under conditions that restrict changes in intracellular Ca2+ and Mg2+ concentrations. The resulting ratio changes for both dyes were reversed completely by the Zn2+ chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, indicating that these dyes are measuring changes in [Zn2+]i. We found that fura-2 was useful in measuring small increases in [Zn2+]i associated with exposure to Zn2+ alone that may be mediated by a Na+/Ca2+ exchanger. Magfura-2, which has a lower affinity for Zn2+, was more useful in measuring larger agonist-stimulated increases in [Zn2+]i. The coapplication of 300 microM Zn2+ and 100 microM glutamate/10 microM glycine resulted in a [Zn2+]i increase that was approximately 40-100 nM in magnitude and could be inhibited by the NMDA receptor antagonist, MK-801 (30 microM), or extracellular Na+. This suggests that Zn2+ influx can occur through at least two different pathways, leading to varying increases in [Zn2+]i. These findings demonstrate the feasibility of measuring changes in [Zn2+]i in neurons.     

The PROP1 2-base pair deletion is a common cause of combined pituitary hormone deficiency

Combined pituitary hormone deficiency (CPHD) has an incidence of approximately 1 in 8000 births. Although the proportion of familial CPHD cases is unknown, about 10% have an affected first degree relative. We have recently reported three mutations in the PROP1 gene that cause CPHD in human subjects. We report here the frequency of one of these mutations, a 301-302delAG deletion in exon 2 of PROP1, in 10 independently ascertained CPHD kindreds and 21 sporadic cases of CPHD from 8 different countries. Our results show that 55% (11 of 20) of PROP1 alleles have the 301-302delAG deletion in familial CPHD cases. Interestingly, although only 12% (5 of 42) of the PROP1 alleles of our 21 sporadic cases were 301-302delAG, the frequency of this allele (in 20 of 21 of the sporadic subjects given TRH stimulation tests) was 50% (3 of 6) and 0% (0 of 34) in the CPHD cases with pituitary and hypothalamic defects, respectively. Using whole genome radiation hybrid analysis, we localized the PROP1 gene to the distal end of chromosome 5q and identified a tightly linked polymorphic marker, D5S408, which can be used in segregation studies. Analysis of this marker in affected subjects with the 301-302delAG deletion suggests that rather than being inherited from a common founder, the 301-302delAG may be a recurring mutation.     

Calcium-dependent and -independent binding of the pentraxin serum amyloid P component to glycosaminoglycans and amyloid proteins: enhanced binding at slightly acid pH

It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) have a stimulatory, and cholesterol 3-sulfate (an amphiphilic anion), an inhibitory, effect on [14C]serine incorporation into phosphatidylserine in glioma C6 and rat liver microsomes. In glioma intact cells sphingosine stimulates phosphatidylserine synthesis in a process independent of protein kinase C, but suppressed by thapsigargin. We suggest that the stimulation of the enzyme occurs by the interaction of amphiphilic cations with the membrane cosubstrate phospholipids, leading to a charge redistribution on their phosphate groups, and hence facilitating the enzyme action. A new hypothesis concerning the mechanism of the serine base exchange reaction is discussed.     

Organization and development of facial motor neurons in the kreisler mutant mouse

The adult facial nerve contains the axons from two populations of efferent neurons. First, the branchiomotor efferent neurons that innervate the muscles of the second arch. These neurons project out of the hindbrain in the motor root and form the facial motor nuclei. Second, the preganglionic efferent neurons that innervate the submandibular and pterygopalatine ganglia. These neurons project from the hindbrain via the intermediate nerve and form the superior salivatory nucleus. The motor neurons of the facial nerve are known to originate within rhombomeres 4 and 5. In the kreisler mouse mutant there is a specific disruption of the hindbrain rhombomeres 5 and 6 appear to be absent. To investigate changes in the organization of the facial motor neurons in this mutant, we have used lipophilic dyes to trace the facial motor components both retrogradely and anterogradely. As expected, facial motor neurons are missing from rhombomere 5 in this mutant. In addition, the loss of these neurons correlates with the specific loss of the superior salivatory nucleus. In contrast, the branchiomeric neurons, that originate in rhombomere 4, appear to develop normally. This includes the caudal migration of their cell bodies forming the genu of the facial nerve. Our studies confirm that rhombomeres are critical to hindbrain development and that they are the fundamental unit at which motor neurons are specified.     

NGF involvement in pain induced by chronic constriction injury of the rat sciatic nerve

Chronic constriction injury (CCI) of the rat sciatic nerve, which within 3 days induces thermal and mechanical hyperalgesia and mechanical allodynia, is used as a model for pain resulting from nerve injury. Involvement of nerve growth factor (NGF) in the development of this hyperalgesia is suggested by the increase in the level of mRNA encoding NGF in cells in the injured area and in dorsal root ganglia at the level of the lesion and the greatly increased NGF levels (determined by ELISA) in the ganglia ipsilateral to the CCI. Application of anti-serum to NGF at the site of CCI delayed the appearance of hyperalgesia, whereas pre-immune serum appeared to enhance it. These results are consistent with the view that NGF is an important factor in the appearance of hyperalgesia associated with unilateral mononeuropathy.     

Dependence of aortic pulse wave assessments on behavioural state in normal term fetus

To investigate any influence of behavioural states on the pulse waves in the descending aorta, 21 human fetuses were studied in utero in uncomplicated gestation at the age of 36-41 weeks. The fetal behavioural states were identified using two real-time scanners and one cardiotocograph. The aortic waveform data and pulse wave velocity (PWV) were recorded by means of two double phase-locking echo-trackers. The PWV of the fetal aorta was significantly lower in fetal behavioural state (FBS) 2F than in FBS 1F, when the fetus was apnoeic, and was also reduced in both states during fetal breathing. The calculated pulse pressure showed the same trend as the PWV. The fetal aortic end diastolic diameter and the pulse amplitude did not alter, when the two states changed. Our data suggest that the central haemodynamics in term fetuses are independently influenced by their behavioural state as well as by fetal breathing. The study of pulse waves in the fetal aorta should preferably be performed during apnoea in state 1F, when neither gross body movements nor breathing movements disturb the recording.     

Absolute metabolite quantification by in vivo NMR spectroscopy: II. A multicentre trial of protocols for in vivo localised proton studies of human brain

We have performed a multicentre trial to assess the performance of three techniques for absolute quantification of cerebral metabolites using in vivo proton nuclear magnetic resonance (NMR). The techniques included were 1) an internal water standard method, 2) an external standard method based on phantom replacement, and 3) a more sophisticated method incorporating elements of both the internal and external standard approaches, together with compartmental analysis of brain water. Only the internal water standard technique could be readily implemented at all participating sites and gave acceptable precision and interlaboratory reproducibility. This method was insensitive to many of the experimental factors affecting the performance of the alternative techniques, including effects related to loading, standing waves and B1 inhomogeneities; and practical issues of phantom positioning, user expertise and examination duration. However, the internal water standard method assumes a value for the concentration of NMR-visible water within the spectroscopic volume of interest. In general, it is necessary to modify this assumed concentration on the basis of the grey matter, white matter and cerebrospinal fluid (CSF) content of the volume, and the NMR-visible water content of the grey and white matter fractions. Combining data from 11 sites, the concentrations of the principal NMR-visible metabolites in the brains of healthy subjects (age range 20-35 years) determined using the internal water standard method were (mean+/-SD): [NAA]=10.0+/-3.4 mM (n=53), [tCho]=1.9+/-1.0 mM (n=51), [Cr + PCr]=6.5+/-3.7 mM (n=51). Evidence of system instability and other sources of error at some participating sites reinforces the need for rigorous quality assurance in quantitative spectroscopy.