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Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by formation of multiple schwannomas and meningiomas due to inactivating mutations in the NF2 tumor suppressor gene on chromosome 22. We describe a polymorphism in the 3' untranslated region of the NF2 gene that is informative in about one-third of individuals. This polymorphism permitted an assessment of the relative expression of NF2 transcripts in lymphoblastoid cell RNA from 22 unrelated NF2 patients heterozygous for a germline NF2 mutation, along with 6 schwannomatosis patients, and 14 unaffected controls. Unequal allelic expression (1.8- to 20-fold) was detected in 15 of the NF2 cases, but in none of the schwannomatosis or control individuals. Underexpression of the NF2 mutant allele was documented for all 6 nonsense or frameshift mutations, 3 of 6 splice mutations, and 1 of 4 missense mutations, which, unexpectedly, was shown to alter the NF2 transcript and create a premature stop codon. In contrast, equal expression or slight overexpression of NF2 mutant alleles was observed for 2 in-frame deletions, 2 splice alterations, and 3 missense mutations. In the remaining 5 cases, the allele representing the mutant transcript was not known. Thus, truncating NF2 mutations, which are the most frequent alterations in NF2 patients and NF2-associated tumors, were associated with underexpression of the mutant allele, whereas the less common in-frame alterations usually showed normal or slight overexpression of the mutant transcript.

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by inactivation of neurofibromin, a protein capable of modulating signal transduction by activating Ras-GTPase activity. We have used cDNA cloning and Northern blot analysis to confirm the NF1 gene produces alternatively polyadenylated mRNAs with 3' untranslated regions (3' UTR) that show striking evolutionary conservation. Scanning of the 3'UTRs for genetic variation revealed three common sequence polymorphisms (> 30% heterozygosity), one less informative polymorphism (approximately 5% heterozygosity) and one rare variant (1/144 chromosomes). These differences were used to examine relative levels of expression of normal and mutant NF1 alleles in lymphoblast cell lines and in one case, autopsy tissue, from patients with NF1. Unequal allelic expression (up to 4-fold) was observed in a subset of both sporadic and familial NF1 cases. Where linkage phase could be determined, the allele segregating with the disorder displayed a relative reduction in expression. However, the magnitude of this effect was variable suggesting the operation of additional, non-genetic factors in determining the degree of relative expression of the mutant allele.

Immunocytochemistry with quantitative image analysis, for both GnRH and its precursor proGnRH-GAP, was used in male European starlings (Sturnus vulgaris) to investigate four stages of a photoperiodically-induced reproductive cycle. Four different groups of birds were examined: photosensitive buy sexually immature, sexually mature, undergoing gonadal regression, and after the completion of regression and fully photorefractory. The size of cells staining for GnRH and proGnRH-GAP increased during gonadal maturation. A reduction in the number of cells staining for GnRH and the size of cells staining for both GnRH and proGnRH-GAP occurred during gonadal regression, though staining for GnRH and proGnRH-GAP in the median eminence remained high at this stage. Birds examined after completion of regression showed significantly reduced staining for both GnRH and its precursor. These observations suggest that photorefractoriness is promoted by a reduction in proGnRH-GAP production and in GnRH synthesis, rather than requiring inhibition of release of GnRH at the median eminence.

Objective: To review the literature on transcutaneous bilirubinometry so that its exact role in the prevention of kernicterus or bilirubin encephalopathy could be determined., Design and Methods: Literature searches were done in Medline and Current Contents., Results: It is estimated that about 50% of newborns have an episode of jaundice in the first few days of life. Six percent of newborns may develop hyperbilirubinemia (> 220 mumol/L), which can potentially cause bilirubin encephalopathy or kernicterus, a severe neonatal disease. In the past, serum bilirubin (SB) has been the preferred method of detecting hyperbilirubinemia in newborns. The ordering of SB in neonates is based on visual evaluation by either physicians or nursing staff. Skin puncture collection of blood exposes the neonate to trauma and risk of infection. A noninvasive device for predicting serum bilirubin levels in newborns diminishes the need to do skin punctures. One such device that has been very extensively studied is the Minolta AirShields Jaundice Meter. It is a portable light-weight instrument that uses reflectance measurements on the skin to determine the amount of yellow color present in the skin, namely transcutaneous bilirubin (TcB). Although the TcB measurements correlate well with serum bilirubin (SB) levels, they cannot accurately predict serum bilirubin because of error related to a variety of factors., Conclusions: TcB cannot be used directly to make decisions about transfusions or phototherapy in neonates. It is a good tool for screening neonates to determine when a laboratory measurement of serum bilirubin is needed. Such a practice requires careful selection of the decision level so that false-negative TcB values do not prevent appropriate serum bilirubin tests from being done.

Objective: To determine what the clinical impact would be of implementing a jaundice meter for use in a busy neonatal service as an adjunctive screening tool for hyperbilirubinemia., Design and Methods: Test utilization data was collected for a 6-month period to determine how neonatal bilirubin was utilized in this hospital. The jaundice meter was evaluated in a study population of healthy term infants. The performance characteristics of the meter and the test utilization data were used to predict the clinical impact a meter could have on screening for hyperbilirubinemia., Results: Utilization data indicated that about 60% of all single bilirubin neonatal testing (i.e., bilirubin only ordered) was done by normal nurseries. A jaundice meter cutoff decision reading of 17 was shown to have a sensitivity of 100% and a specificity of 68% for hyperbilirubinemia (> 260 mumol/L) in a study population of healthy term infants. From this data, it was estimated that use of a jaundice meter could eliminate 43% of the single (i.e., not combined with other tests) bilirubin tests done on healthy term neonates with no prior exposure to phototherapy. This constitutes an overall 20% reduction in bilirubin testing in normal nurseries when testing done on babies exposed to phototherapy and combined bilirubin testing are taken into consideration. Additionally, it was shown that there would be an improvement of 9% in the prediction of hyperbilirubinemia without loss of 100% sensitivity., Conclusion: Use of a jaundice meter in normal nurseries as an adjunctive screening tool enhances patient care by reducing the overall blood procurement rate in normal nurseries by 20% and increasing screening efficiency for significant hyperbilirubinemia by 5%.

Neurofibromatosis 2 (NF2) is an autosomal dominant disease characterized by bilateral vestibular schwannomas and other nonmalignant tumors of the brain, spinal cord, and peripheral nerves. Although the average age of onset of NF2 is 20 years, some individuals may become symptomatic in childhood. We studied 5 unrelated NF2 patients who became symptomatic before age 13. All 5 had multiple tumors in addition to vestibular schwannoma, and none had a positive family history. Sequence analysis of the NF2 gene revealed identical nonsense mutation of exon 6 in 3 patients. Because this mutation destroys a restriction enzyme recognition site, genomic DNA from the 2 other children was directly tested for this change and identical alterations were detected. Although the work of our laboratory and others has not, in general, detected identical mutations in unrelated patients, this mutation seems to occur particularly frequently in the pediatric population and thus may be associated with an especially severe phenotype. Restriction analysis in children with NF2 may be a cost effective way of identifying their mutation. Further work is needed to characterize the effects of this change on the NF2 protein product and its relationship to this severe phenotype.

Neurofibromatosis 2 (NF2) features bilateral vestibular schwannomas, other benign neural tumors, and cataracts. Patients in some families develop many tumors at an early age and have rapid clinical progression, whereas in other families, patients may not have symptoms until much later and vestibular schwannomas may be the only tumors. The NF2 gene has been cloned from chromosome 22q; most identified germ-line mutations result in a truncated protein and severe NF2. To look for additional mutations and clinical correlations, we used SSCP analysis to screen DNA from 32 unrelated patients. We identified 20 different mutations in 21 patients (66%): 10 nonsense mutations, 2 frameshifts, 7 splice-site mutations, and 1 large in-frame deletion. Clinical information on 47 patients from the 21 families included ages at onset and at diagnosis, numbers of meningiomas, spinal and skin tumors, and presence of cataracts and retinal abnormalities. We compared clinical findings in patients with nonsense or frameshift mutations to those with splice-site mutations. When each patient was considered as an independent random event, the two groups differed (P < or = .05) for nearly every variable. Patients with nonsense or frameshift mutations were younger at onset and at diagnosis and had a higher frequency and mean number of tumors, supporting the correlation between nonsense and frameshift mutations and severe NF2. When each family was considered as an independent random event, statistically significant differences between the two groups were observed only for mean ages at onset and at diagnosis. A larger data set is needed to resolve these discrepancies. We observed retinal hamartomas and/or epiretinal membranes in nine patients from five families with four different nonsense mutations. This finding, which may represent a new genotype-phenotype correlation, merits further study.

Retrograde transport of fluorescent latex microspheres was combined with immunocytochemistry for glutamate to determine the organization of the projections from glutamate-containing neurones in the rostral hypothalamus to the different subdivisions of the periaqueductal grey (PAG). Double-labelled neurones, i.e. neurones immunoreactive for glutamate and projecting to the PAG, were found throughout the rostral hypothalamus. There were no apparent differences, however, in the origins of presumed glutamatergic projections from the rostral hypothalamus to the different subdivisions of the PAG.

Hereditary lymphedemas that are not associated with other malformations usually affect the lower limbs and are inherited in an autosomal dominant fashion. These non-syndromic hereditary lymphedemas are categorized by their age of onset, being either congenital (Milroy disease) or having an onset in childhood or around puberty (Meige disease). We describe a family in which three individuals in three generations had unusually late onset of lymphedema in their mid-twenties or thirties. The proband additionally developed a very rare lymphangiosarcoma. This tumor, usually associated with post-mastectomy lymphedema, has not been described in late-onset hereditary lymphedema. Because of an unusually high incidence of multiple primary tumors in association with lymphangiosarcoma in the literature (approximately 10%) and the proband's own familial cancer background, we speculate that an inherited predisposition to malignancy may underlie the development of lymphedema-associated lymphangiosarcoma.

Retinoblastoma (Rb) is a rare childhood tumor of the eye. In the heritable form, tumors are often bilateral and survivors have a greatly increased risk both for a second malignancy and for having children with Rb. Familial patterns of both cancer and birth defects are poorly understood in families with a heritable cancer, and little is known of the ways that a heritable cancer affects the lives of long-term survivors. To find out more about these and other issues in the lives of long-term survivors of childhood and adolescent cancer, we interviewed 56 adult survivors of retinoblastoma (15 with the heritable form) and 84 brothers and sisters as controls, who formed part of a large retrospective cohort study. Rb survivors were interviewed between 1980 and 1983, when they were 30 years old on average. Types of employment and health problems did not differ between survivors and controls, regardless of sight, but the income of blind survivors was considerably less than that of partially sighted survivors. Despite similar marriage rates, fewer survivors than controls reported a pregnancy (RR = 0.45; 95% CI; 0.24-0.83 for both sexes combined). Parents of children with heritable Rb seemed more likely to have had cancer than parents in families with nonheritable Rb (P = 0.06), and mothers were more likely than fathers to be affected (P = 0.01). This small series suggests that having retinoblastoma may have many long-term consequences, reaching beyond genetic and physical effects to touch family life and income attainment and the health of other family members. Follow-up of more modern cohorts and the use of molecular tools will clarify the long-term consequences of more recent therapies, and patterns of familiar cancer.

To determine the spectrum of manifestations in neurofibromatosis 2 (NF2) and to assess possible heterogeneity, we evaluated 63 affected individuals from 32 families. Work-up included skin and neurologic examinations, audiometry, a complete ophthalmology examination with slit-lamp biomicroscopy of the lens and fundus, and gadolinium-enhanced MRI of the brain and, in some, of the spine. Mean age-at-onset in 58 individuals was 20.3 years; initial symptoms resulted from vestibular schwannomas (44.4%), other CNS tumors (22.2%), skin tumors (12.7%), and ocular manifestations including cataracts and retinal hamartomas (12.7%). Five asymptomatic individuals were diagnosed through screening. Vestibular schwannomas were documented in 62 individuals (98.4%); other findings included cataracts (81.0%), skin tumors (67.7%), spinal tumors (67.4%), and meningiomas (49.2%). Usually, clinical manifestations and course were similar within families but differed among families. To assess possible heterogeneity, we assigned affected individuals to three proposed subtypes (representing mild, intermediate, and severe NF2) based on age-at-onset, presence or absence of CNS tumors other than vestibular schwannomas, and presence or absence of retinal hamartomas. Comparisons among the three subtypes for many clinical parameters demonstrated that patients in the mild subtype differed from those in the other two subtypes for most parameters, but that none of the parameters distinguished patients in the intermediate subtype from those in the severe subtype. Thus, there are likely two rather than three subtypes of NF2. Classification of patients to subtype may aid in counseling about long-term prognosis and in formulating individualized guidelines for medical surveillance.

To investigate the hypothesis that persons who developed thyroid or skin cancer subsequent to scalp irradiation for tinea capitis are particularly sensitive to radiation, possibly because of a high frequency of ataxia-telangiectasia, we used an in vitro cell survival assay to evaluate radiosensitivity of their fibroblast cell strains. Study subjects were selected from a cohort of 10,834 Israelis irradiated during childhood for tinea capitis. Skin fibroblasts were obtained from thyroid and skin cancer patients (cases) as well as a sample of subjects who did not have cancer (controls). Fibroblasts were cultured and then loss of colony-forming ability as a result of acute X-irradiation was evaluated. Comparison of survival curve parameters (mean inverse of the slope and the dose needed to reduce colony survival to 10%) between 12 thyroid cancer and 12 control strains showed no differences (P > 0.5). A slightly increased radiation sensitivity of the skin cancer cases compared with their controls was observed. Although based on few subjects (14 cases and 11 controls), the findings were similar whether the mean inverse of the slope (P = 0.06) or the dose needed to reduce colony survival to 10% (P = 0.05) was evaluated. However, because of the small size of the study and potential errors inherent in survival assays, our finding that cell strains derived from patients who developed skin cancer exhibit enhanced radiosensitivity should be viewed as preliminary and interpreted cautiously.

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by multiple tumors of the central nervous system, predominantly bilateral vestibular schwannomas. The gene for NF2 is located in the chromosomal region 22q12 between the loci D22S1 and D22S28. We have performed genetic linkage analysis on 13 NF2 families with a total of nine polymorphic DNA markers, including five which we have recently mapped to this region. Two loci, D22S32 and NEFH, are linked to the NF2 locus at 0% recombination (lod scores of 6.03 and 4.28, respectively). By multipoint linkage analysis, we assign the NF2 gene to an interval of 7 cM, between the loci D22S212 and D22S28. We have used this set of nine markers to construct chromosome 22 haplotypes for the 82 at-risk individuals in this pedigree set. It has been possible to determine, with a high degree of certainty, the carrier status of 70 (85%) of these at-risk individuals. Risk prediction was possible in every case where DNA was available from both parents. Fifty-three of the 70 (76%) informative individuals were assigned decreased risks of being carriers. The use of chromosome 22 probes for risk assessment should result in a greatly reduced number of individuals who require periodic screening for NF2.

Neural input to the hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons was investigated in male starlings (Sturnus vulgaris) using electron microscopy combined with immunocytochemistry. Birds (4 to 6 in each group) were sampled at four stages of a photoperiodically induced reproductive cycle: (a) sexually immature but photosensitive, under short days; (b) during sexual maturation after 7 to 25 long days; (c) during gonadal regression after 35 to 50 long days; and (d) when fully photorefractory after 11 months exposure to long days. The length of the perikaryal membrane, the number and length of axo-somatic terminals in contact with it and the number and length of synaptic modifications within the terminals were measured for a minimum of six LHRH neurons in each brain. The number of axo-somatic terminals per neuron and the number per unit length of perikaryal membrane did not differ in birds of groups (a), (b) and (c), but was significantly greater (P < 0.05) in the fully refractory birds (group d). Similarly, the number of synaptic modifications was significantly greater (P < 0.05) in group (d) than in the other groups. These results are consistent with increased neural input to the LHRH perikarya in photorefractory birds after prolonged exposure to long days, although there was no indication of a change in input at the time of gonadal regression.

This prospective study of 96 individuals from 29 families with neurofibromatosis 2, 49 of whom were affected, confirms in an extended series the previously reported association between posterior subcapsular/capsular cataract and neurofibromatosis 2. Posterior subcapsular/capsular cataracts were found in 36 (80%) of the 45 affected individuals (four individuals were excluded from statistical analyses). In addition, the association of peripheral cortical lens opacities with neurofibromatosis 2 was found to be statistically significant. Seventeen of the patients with neurofibromatosis 2 (37.8%) had peripheral cortical cataracts in comparison with none of the unaffected family members (p < 0.0001). In three patients peripheral cortical opacities were present despite the absence of posterior subcapsular/capsular cataracts. These findings support the inclusion of cortical cataracts of early onset, in addition to posterior subcapsular/capsular cataracts, in the diagnostic criteria of neurofibromatosis 2.

Neurofibromatosis type 2 (NF2) is an autosomal dominant syndrome characterized by the development of vestibular schwannomas and other tumors of the nervous system, including cranial and spinal meningiomas, schwannomas, and ependymomas. The presence of bilateral vestibular schwannomas is sufficient for the diagnosis. Skin manifestations are less common than in neurofibromatosis type 1 (NF1; von Recklinghausen disease). The apparent clinical distinction between NF1 and NF2 has been confirmed at the level of the gene locus by linkage studies; the gene for NF1 maps to chromosome 17, whereas the gene for NF2 has been assigned (in a single family) to chromosome 22. To increase the precision of the genetic mapping of NF2 and to determine whether additional susceptibility loci exist, we have performed linkage analysis on 12 families with NF2 by using four polymorphic markers from chromosome 22 and a marker at the NF1 locus on chromosome 17. Our results confirm the assignment of the gene for NF2 to chromosome 22 and do not support the hypothesis of genetic heterogeneity. We believe that chromosome 22 markers can now be used for presymptomatic diagnosis in selected families. The NF2 gene is tightly linked to the D22S32 locus (maximum lod score 4.12; recombination fraction 0). A CA-repeat polymorphism at the CRYB2 locus was the most informative marker in our families (lod score 5.99), but because the observed recombination fraction between NF2 and CRYB2 was 10 cM, predictions using this marker will need to be interpreted with caution.

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude "mitochondrial" fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude "mitochondrial fraction," a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to nonmitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.

Combined pigment epithelial and retinal hamartomas are rare lesions that usually occur sporadically in individuals without systemic abnormalities. However, they have been reported in isolated patients with neurofibromatosis 1 and 2. No familial cases have been reported. The cases of four patients with unilateral macular lesions from three consecutive generations of a single family are presented: two of the patients also have neurofibromatosis 2. The ophthalmoscopic appearance of their ocular lesions resembles combined pigment epithelial and retinal hamartomas. The morphologic differences in the lesions of these 4 patients, whose ages are 8 months, 5 years, 29 years, and 65 years, may serve to demonstrate the evolution of this type of hamartoma.

Occipitofrontal circumference (OFC) was strongly correlated with height in 72 normal Caucasian men (r = 0.28, P = 0.018) and 78 women (r = 0.53, P less than 0.0001). OFC:height ratios were approximately normally distributed in each sex with a mean of 0.326 (standard deviation [sd] = 0.0139) in males and 0.335 (sd = 0.0177) in females. Relative macrocephaly (an OFC greater than the 95th centile for height) was seen in seven of nine probands with the nevoid basal cell carcinoma syndrome (NBCC), eight of 32 non-probands with NBCC, three of four neurofibromatosis type 1 (NF1) probands, and six of nine non-probands with NF1. Thirty-one percent of the non-proband NBCC cases had OFC less than the 50th centile for height, while none of the NF1 cases had this finding. Head size appears to be related to proband status in NBCC, while the evidence suggests that NF1 is a true macrocephaly syndrome. Objective detection of relative abnormalities in head size may aid in syndrome delineation and diagnosis.

The neurofibromatoses comprise at least two autosomal dominant disorders affecting an estimated 100,000 Americans with clinical manifestations that may require care from every type of clinician. Neurofibromatosis 1 and neurofibromatosis 2 have in common the occurrence of many neurofibromas but are distinctly different clinical disorders. The disease genes are on different chromosomes. Magnetic resonance imaging, particularly with gadolinium enhancement, has generally supplanted other techniques for visualizing brain, spinal, and other neural tumors in both disorders. The technique has rekindled the controversy over the nature and frequency of optic pathway tumors in patients with neurofibromatosis 1 and has revealed, throughout the brains of young patients, bright lesions that have uncertain clinical consequences and unknown pathologic bases. In patients with neurofibromatosis 2, small acoustic neuromas can be seen, leading to the possibility of excision with preservation of hearing and facial nerve function. Abnormal hearing may occur to excess in patients with neurofibromatosis 1, but acoustic neuroma has never been documented. In patients with neurofibromatosis 2, a battery of audiologic tests has a high positive predictive power. Lisch nodules or iris hamartomas, probably a universal sign in adults with the neurofibromatosis 1 gene, cause no problem with vision. Posterior capsular lens opacity in patients with neurofibromatosis 2 is a helpful diagnostic sign and a potential source of additional handicap in persons at risk for impaired hearing. Progress in the clinical delineation of the disorders has been matched with considerable research into the still obscure pathogenesis of the disorders. Such rapid advances may necessitate reconsideration of the conclusions of the National Institutes of Health Consensus Development Conference on Neurofibromatosis, especially those on the categories of persons in which a neurofibromatosis should be considered and the need for caution in recommending surgery. Watchful waiting may often be the best management for acoustic neuromas in neurofibromatosis 2.

A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.

The autoradiographic localization of radiolabelled taurine taken up in the rat substantia nigra in vivo together with conditions of release of the [3H]taurine taken up into brain slices were studied to determine whether they are consistent with the hypothesis that taurine may act as a neurotransmitter in the striatonigral pathway. At the light microscopic level the main cellular elements that became radiolabelled following the injection of [3H]taurine into the substantia nigra could be identified as glial cells. Electron microscope autoradiography confirmed that a subpopulation of glial cells including astrocytes, pericytes, and oligodendrocytes were radiolabelled and that neuronal perikarya were not radiolabelled. In addition, axonal elements including both terminal and preterminal boutons were found to have silver grains overlying them and were thus considered to be radiolabelled. This was supported by a quantitative analysis of the distribution of the silver grains; whereas glial elements had a significantly higher number of grains associated with them than with any other structure, axonal elements had a significantly greater number of grains than dendritic structures. Release of the preloaded [3H]taurine from superfused slices of substantia nigra occurred in response to veratridine, was calcium-dependent and was sensitive to inhibition by high magnesium concentrations or tetrodotoxin. Following the destruction of neurons in the striatum by ibotenic acid injections, although the weight of the ipsilateral substantia nigra was reduced, the uptake of [3H]taurine was not altered. In contrast to this, the veratridine-stimulated release was markedly attenuated, implying that the destruction of striatal neurons causes the loss of sites in the substantia nigra from which exogenous taurine is released. These results add further support to previous suggestions that taurine might act as a neurotransmitter or neuromodulator in the striatonigral pathway.

Three new families are reported for standard gene linkage markers and classical peripheral neurofibromatosis (Von Reckling-hausen disease). Additional data are summarised for the exclusion map. One family gives slight evidence of close linkage with the Gc locus on chromosome 4, raising again the question of possible genetic heterogeneity in NF.

The proxidase anti-peroxidase immunocytochemical staining technique has been used to identify prolactin and growth hormone cells in pituitaries from fetal and neonatal sheep. The size of the secretory granules in these cell types has been measured using the image analysing computer Quantimet 720. The area size distributions of the fetal prolactin and growth hormone granules were compared with those in the neonate and the adult. It appears that the gestional age of the fetus may influence the size range of prolactin secretory granules.

In many birds reproduction is triggered by long daylengths but, paradoxically, continued exposure to long days leads to photorefractoriness and a complete shut down of the reproductive system. As these effects are thought to be mediated through the secretion of LH-RH, immunocytochemical techniques were used to investigate changes in the LH-RH system when European starlings were exposed to different photoperiods. Starlings exposed to 11L:13D and with mature testes show strong immunostaining both of LH-RH perikarya and fibers. Photosensitive short-day (8L:16D) starlings with undeveloped testes show an almost identical distribution of strongly immunoreactive perikarya but with less dense fibre staining. However, long-day (18L:6D) photorefractory starlings with fully regressed testes, show a profound reduction in LH-RH immunostaining. Perikarya have the same distribution but show a much reduced intensity of staining and fibers had almost entirely disappeared from all regions of the brain. Preliminary observations on the ultrastructure of immunocytochemically identified LH-RH neurones are also reported.

A 32-year-old black man was observed with osteosarcoma and multiple anomalies including deafness, hypopigmentation, cataracts, small head size, hypogonadism, and restricted joint mobility. The birth defects may comprise a new syndrome or combination of syndromes, of which the malignancy may be a part.

The subcellular location of hexokinase was investigated in rat kidney. Both soluble and particulate locations are indicated by differential centrifugation. The particulate form is predominant, representing about 80% of the total activity. None of the activity is latent. Density gradient centrifugation followed by marker enzyme analysis reveals the presence of two populations of mitochondria with distinct densities. Hexokinase is associated primarily with the mitochondrial population having the lower density. Association of hexokinase with brush border, plasma membrane, lysosomes, and endoplasmic reticulum is considered unlikely on the basis of density gradient centrifugation and enzyme analysis. About 95% of the hexokinase activity associated with the mitochondrial fraction can be released in soluble form by repeated incubations with glucose 6-phosphate. An incubation time of about 4 min at 30 degrees C is required to achieve a maximal solubilizing effect. Release is accomplished without disrupting the mitochondrial compartments. Hexokinase is released also by treatment of the mitochondrial fraction with increasing concentrations of digitonin. This technique disrupts and differentially releases the mitochondrial compartments. As observed with liver, but in contrast to that observed with tumor (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912), the release of hexokinase from the mitochondrial fraction of kidney does not correlate with the release of enzymes known to mark the mitochondrial membranes or compartments. These studies provide the first critical evidence about the subcellular location of hexokinase in kidney. They show that in this tissue hexokinase is associated primarily with low density mitochondria, a finding that adds credibility to the existence of this discrete population of mitochondria in vivo. Significantly, this association of hexokinase with kidney mitochondria appears unique in that its release on submitochondrial fractionation does not correlate with the release of known mitochondrial marker enzymes. These results are directly relevant to those cells in the kidney which utilize glucose as an energy source. It is suggested that the enhanced glycolytic capacity of these cells may be due, at least in part, to an association of hexokinase with low density mitochondria.

Using the peroxidase-antiperoxidase immunocytochemical staining technique, prolactin and growth hormone cells have been identified and described in the ovine pituitary. The image analysing computer, Quantimet 720, was used to assess accurately the size range of the secretory granules in these cell types. The area size distributions of the prolactin and growth hormone granules are similar. An increased proportion of larger granules was observed in the prolactin cells post-partum. Serial sections stained alternately for prolactin or growth hormone confirmed that the cells contain either prolactin or growth hormone but not both.

The activity of phosphoenolpyruvate carboxykinase (PEPCK) increased in the rat kidney after induction of metabolic acidosis. Immunotitration of the enzyme in renal extracts from normal and acidotic rats indicate that this increase in catalytic activity correlates with the amount of immunotitrateable activity. PEPCK activity decreased rapidly during recovery from acidosis and the time required for the activity to return to normal was not altered by the administration of NaHCO3 at the start of recovery. Immunotitration of the enzyme from rats allowed to recover from metabolic acidosis revealed that the decrease in catalytic activity was paralleled by a decrease in the amount of immunotitrateable activity. It is concluded that the rise and fall in PEPCK activity during acidosis and recovery from acidosis, respectively, are due to comparable changes in enzyme protein.

The influence of protein intake on acid excretion and renal glutamine metabolism was investigated and compared to the effects of NH4Cl-induced metabolic acidosis. Rats fed a diet containing 55% casein excreted more ammonia, phosphate, sulphate, and chloride than did rats fed a 13% casein diet, but, when they were given an 0.1 M NaHCO3 solution to drink, ammonia excretion was no longer elevated. Renal phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase activities, ammoniagenesis by isolated mitochondria, and the rate of renal gluconeogenesis were all elevated in the rats fed the high-protein diet but not if these rats also drank the sodium bicarbonate solution. Increased glutaminase and phosphoenolpyruvate carboxykinase activities, mitochondrial ammoniagenesis, and gluconeogenesis were all evident in rats made acidotic with NH4Cl. It is concluded that these metabolic adaptations evident in the kidneys of rats fed the high-protein diet are due to the acidogenic effects of increased protein intake.

An improved method for the analysis of high resolution electron microscope autoradiographs is described which is simple and quick to perform. The method uses a transparent screen superimposed over the autoradiograph prints to provide information on the distribution of grains over neighboring structures, produced by radioactive disintegrations occurring in different regions of the section. This "cross-scatter" information is used to estimate the radioactivity in different structures without the need to make any assumptions about the size, shape and arrangement of the structures within the autoradiographs being analyzed. The method allows activity in membranes and other linear structures to be determined and also the accuracy attributable to the activity values obtained.

In addition to reporting, in accompanying papers, their individual analyses of mapping the neurofibromatosis type 1 (NF1) gene on chromosome 17, members of the International Consortium for NF1 Linkage contributed their data for our joint analysis to determine the exact sequence of flanking markers and to obtain precise estimates and confidence limits of the recombination fractions for the closest markers, in anticipation of clinical use. With specimens from 142 families and more than 700 affected persons, eight teams used 31 markers in the pericentric region of chromosome 17 to perform 13,838 genotypings. With the combined data, we used the computer program CRI-MAP to build the most likely sequence of loci by sequentially adding single loci to a fixed pair of loci and separately calculating the likelihood of all permutations of four consecutive loci. The best order is pter-pA10-41-EW301-centromere (p17H8)-pHHH202-NF1-EW206-EW207-EW203++ +-CRI-L581-CRI-L946-HOX2-NGFR-qter. The total genetic distance from pA10-41 to NGFR is 26 cM in males and 56 cM in females, and the overall difference in sex-specific maps is statistically significant (P = .006). The upper 99% confidence limits of the recombination fraction of the closest proximal marker, pHHH202, is 4%, and that for the closest distal marker, EW206, is 9%. These limits should decrease with the use of additional probes and the further evaluation of DNA from the six persons showing multiple recombinations within short genetic distances. Clinical application is technically feasible with currently available markers, although its appropriate use for prenatal and presymptomatic diagnosis requires further discussion and evaluation.

An autosomal euchromatic maternal-effect mutant, abo (= abnormal oocyte), interacts with, or regulates the activity of, the heterochromatin of the sex chromosomes of Drosophila melanogaster. It is shown that this interaction or regulation with the X chromosome involves a specific heterochromatic locus or small region that maps to the distal penultimate one-eighth of the basal X-chromosome heterochromatic segment.

Genetic factors in the etiology of carotid body tumors (CBT) were sought in a medical record review of 222 histologically diagnosed cases at 12 U.S. medical centers. Patients in the series, which had a marked female predominance (146 females:76 males), usually developed tumors between the fourth and seventh decades of life (mean, 44.7 yr). In 16 patients who also had other extra-adrenal paragangliomas, suggesting a multiple primary tumor syndrome, CBT were diagnosed significantly earlier (mean, 35.4 yr; P less than 0.01). The occurrence of thyroid cancer in 5 other patients appeared to be excessive. Familial CBT was recognized in 16 patients from 13 affected families, including 9 newly ascertained kindreds. Compared with non-familial lesions, familial CBT tended to develop bilaterally (38% vs. 8% unilaterally) and at slightly earlier ages (41.6 vs. 44.9 yr). CBT was reported to occur in an autosomal dominant pattern in some families and within sibships in others; relatives were not examined for confirmation. The familial findings are generally consistent with a two-step mutation model of the development of hereditary and nonhereditary CBT; apparent deviations from the model might be clarified with additional data on this rare neoplasm.

Immunocytochemistry was used to determine if photoperiod and/or sex have any effect on the pattern of the luteinizing hormone-releasing hormone (LHRH) system in the brain of the Japanese quail. Immunopositive perikarya were found within three major areas of the brain: the rostral paraolfactory lobe, the preoptic, and the septal region. A quantitative analysis of LHRH cell numbers was performed on male and female quail after two photoperiodic treatments: sexually mature birds exposed to 24 weeks of 20 h light: 4 h darkness (20L:4D), and birds with a regressed reproductive system (induced by transfer from a photoregime of 20L:4D to 25 short days of 8L:16D). Two-way analysis of variance showed that short-day males display significantly (p less than 0.05) more immunopositive perikarya (607 +/- 134) than long-day males (291 +/- 114), short-day females (293 +/- 103) or long-day females (330 +/- 92). The density of LHRH-immunoreactive nerve fibres and the intensity of the immunostaining in the median eminence were always greater in long-day sexually mature quail (male and female) than in animals exposed to 25 days of 8L:16D. These results demonstrate that the LHRH system of the quail is influenced by photoperiod and mirrors sexual differentiation.

Baseline and mutagen-induced levels of sister-chromatid exchanges were evaluated in 10 normal individuals. Cultures with whole blood or purified lymphocytes, either freshly isolated or after 1 or 6 months of cryopreservation, were analyzed to determine whether frozen lymphocytes are suitable for SCE studies. Whole blood and freshly isolated lymphocytes were cultured from samples taken at the beginning of the study (Time 0) and 6 months later (Time 6). Cryopreserved lymphocytes were recovered after 1 month (Time 1) and 6 months (Time 6) of cryopreservation and then challenged with mutagens in culture. The mutagens used were mitomycin C, 4-nitroquinoline-1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine. Purified lymphocytes had consistently and significantly higher baseline SCE frequencies than cells from whole blood cultures and were more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and 4-nitroquinoline-1-oxide. The response to mitomycin C was similar in all culture types. There was, overall, no consistent effect of freezing on baseline or induced sister-chromatid exchange frequencies in the purified lymphocytes. This suggests that purification and cryopreservation of human lymphocytes does not alter the baseline or mutagen-induced sister-chromatid exchange response and in certain epidemiological, occupational and monitoring situations may have logistical and technical advantages over the use of fresh whole blood.

Two middle age sisters had most manifestations of the SC phocomelia syndrome including postnatal growth retardation, symmetric limb deficiencies with radial aplasia and absent thumbs, facial anomalies with microcephaly, microphthalmia, hypoplastic nasal alae, and borderline to mild mental retardation. Unusual findings included congenital paralysis of some cranial nerves in both patients and malignant melanoma in the proposita. Cultured lymphocytes from both patients, and skin fibroblasts, Epstein Barr virus-transformed lymphocytes, and tumor cells from the proposita demonstrated premature separation of centromeric heterochromatin (PCS) of many chromosomes, a finding noted previously in the SC phocomelia syndrome and the similar but more severe Roberts syndrome. Extensive overlap of the phenotypes of the sisters and 15 other patients with either syndrome and PCS confirms that these are either allelic conditions or the same disease--designated Roberts-SC phocomelia syndrome. The role of PCS in the syndrome(s) remains uncertain since some patients with the characteristic clinical phenotypes are reported to lack it.

The development of the reproductive system was studied in juvenile starlings during the acquisition of photosensitivity, the attainment of sexual maturation after photostimulation and the subsequent onset of photorefractoriness, using immunohistochemistry for LHRH and radioimmunoassay measurements of hypothalamic, pituitary and plasma hormone concentrations. The first stage of sexual development induced by exposure of photorefractory immature starlings to short days (8 h light:16 h darkness; 8L:16D) was characterized by a decrease in pituitary prolactin content within 1 week and an increase in hypothalamic LHRH content, in the size of the LHRH perikarya and in the intensity of immunostaining in the median eminence in 4-6 weeks. Sexual maturation occurring after exposure to long days (18L:6D) was associated with further increases in LHRH content and cell size, and increases in LH and prolactin concentrations. During testicular regression, LHRH perikarya were reduced in size and staining intensity but LHRH immunostaining in the median eminence and content in the hypothalamus remained high until gonadal regression was almost complete. Prolactin levels were maximal during testicular regression. These results suggest that gonadal regression is initiated by a reduction in LHRH synthesis and possibly, in addition, an external inhibitory influence on LHRH release. Hypothalamic LHRH content eventually declined and LHRH immunostaining in the median eminence was much reduced in fully photorefractory starlings maintained under long days.

A detailed investigation concerned with localizing hexokinase in the Novikoff ascites tumor is presented. At least 50% of the total hexokinase activity was shown by differential and density gradient centrifugation techniques to be associated with tumor mitochondria. None of this activity was latent. Fractionation of isolated tumor mitochondria with digitonin revealed an outer membrane location for this enzyme. Treatment of tumor mitochondria with glucose 6-phosphate released about 80 to 85% of the hexokinase activity without disrupting the intermembrane compartment. This suggests that at least this proportion of the activity is bound to the outer surface of the outer membrane. Successive treatments did not remove the remaining hexokinase activity. At 30 degrees C, an incubation time of about 10 min with glucose 6-phosphate was required to achieve maximal release. No solubilization occurred at 0-4 degrees C. The isozymes derived from Novikoff mitochondria were identified by anion exchange chromatography as types I and III. Glucokinase activity was not detectable. Evidence is also presented which indicates that the hexokinase obtained from Novikoff mitochondria binds to the outer membrane of rat liver mitochondria. In contrast, the low endogenous hexokinase activity present in isolated liver mitochondria was found not to fractionate with outer membrane markers, but rather with contaminating microsomal membrane markers. Results described here provide the first direct evidence for the submitochondrial location of hexokinase in a tumor. They reveal an outer membrane location and an involvement of two hexokinase isozymes. Because these findings are characteristic of the hepatoma and not observed in control liver preparations, it is suggested that they may be very relevant to the general property of rapidly growing tumors to catabolize large amounts of glucose.

A translocation that involves chromosome X (band p11.2) and chromosome 18 (band q11.2) was observed in short-term in vitro cultures of cells from five synovial sarcomas and one malignant fibrous histiocytoma. In four of these tumors, the translocation t(X;18)(p11.2;q11.2) was reciprocal. The two other tumors had complex translocations: t(X;18;21)(p11.2;q11.2;p13) and t(X;15;18)(p11.2;q23;q11.2). A translocation between chromosomes X and 18 was not detected in other histological types of soft tissue sarcoma. The X;18 rearrangement appears to characterize the synovial sarcoma and is the first description of a primary, nonrandom change in the sex chromosome of a human solid tumor.

Different cell types from a female patient with Roberts/SC phocomelia syndrome were evaluated quantitatively for the presence of repulsion of heterochromatin and satellite regions of mitotic chromosomes. Whereas EBV-transformed lymphoblasts from an established cell line revealed these phenomena at frequencies equal to those in PHA-stimulated lymphocytes and cultured skin fibroblasts, aneuploid cells from a metastatic melanoma displayed them at 50% lower frequency. Cocultivation of the patient's fibroblasts with either an immortal Chinese hamster cell line or with a human male fibroblast strain carrying a t(4;6)(p14;q21) translocation showed that the phenomenon was not corrected or induced by a diffusible factor or by cell-to-cell contact. In each experiment, only the patient's metaphase spreads revealed chromatid repulsion. In fusion hybrids between the patient's fibroblasts and an established Chinese hamster cell line, the human chromosomes behaved perfectly normally, suggesting that the gene product which is missing or mutant in Roberts/SC phocomelia syndrome is supplied by the Chinese hamster genome.