Molecular characterization of a patient with del(1)(q23–q25)

Summary We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23–1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23–q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000–16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21–1q25.     

Influence of DNA sequence on the nature of mispairing during DNA synthesis

A series of synthetic oligonucleotide primers, annealed at various positions along the lacZ-alpha region of bacteriophage M13mp9 template, were elongated by purified DNA polymerases in the presence of only 3 of the 4 deoxynucleoside triphosphates to achieve misincorporation at a total of 49 different positions along the template. The newly synthesized strands (containing misincorporated bases) were isolated and sequenced to determine the identity of misincorporated deoxynucleoside monophosphates. The results indicate that the kind of mispairing that occurs during DNA synthesis is greatly influenced by the nucleotide sequence of the template. Transition-type base substitutions predominated overall, but at many template positions, transversion-type base substitutions occurred, most commonly via A.A mispairing. The results of parallel determinations made with Escherichia coli DNA polymerase I ("large fragment" form) and DNA polymerase of Maloney murine leukemia virus indicated that, overall, the identity of polymerase had only a small effect on the kind of misincorporation that occurred at different positions along the template. However, at certain template positions, the nature of mispairing during DNA synthesis was reproducibly affected by differing polymerase active-site environment.     

Aggressive chemotherapy in adult primary myelodysplastic syndromes. A report on 29 cases

Twenty-nine adult patients with primary myelodysplastic syndromes (MDS) and an excess of marrow blasts were treated by aggressive chemotherapy while still in MDS phase (20 cases) or after progression to ANLL (9 cases). Median age was 47.5 (range 18-68). Twenty-eight patients received a combination of Rubidazone and Ara C and 1 received High dose Ara C. Fourteen patients (48%) achieved complete remission (CR), 5 (17%) were treatment failures (F) and 10 (35%) died during therapy induced aplasia (DA). Median disease free survival was 8.5 months. Median survival of the whole population was 6 months from the onset of treatment, and 17 months in patients achieving CR. These results were significantly less favorable than those obtained at our institution in de novo ANLL with the same chemotherapy regimens. No statistically significant prognostic factors of treatment outcome emerged but patients with normal cytogenetic findings seemed to have both a higher CR rate and longer remissions than patients with abnormal karyotypes. Patients under 50 did not have higher CR rates than older patients, although they had longer remissions (with 3 out of 6 CRs exceeding 2 years). Finally, treatment outcome and survival were identical in patients treated in the MDS phase and in those treated after progression to ANLL. Combination chemotherapy is a highly toxic approach in MDS and essentially seems to benefit younger patients with a normal karyotype, in whom some long remissions can be obtained.     

Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots

The subunit organization of the tonoplast H+-pumping ATPase from oat roots (Avena sativa L. var. Lang) was investigated. Tonoplast vesicles were treated with low ionic strength solutions (0.1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer or 0.1 mM Na EDTA), carbonate, or a chaotropic reagent (KI), and then centrifuged to give a soluble fraction and a pellet. Treatments with low ionic strength solutions or KI resulted in 70-80% reduction in the membrane-associated ATPase activity, but did not affect the K+-stimulated pyrophosphatase activity. Polypeptides of 72, 60, and 41 kDa were solubilized from tonoplast vesicles by these wash treatments. These polypeptides reacted with polyclonal antibodies against the holoenzyme of tonoplast ATPase (anti-ATPase) and copurified with the tonoplast ATPase activity during gel filtration chromatography (Sepharose CL-6B). Mono-specific antibody against the 72- or 60-kDa polypeptide reacted with the solubilized 72- or 60-kDa polypeptide, respectively. However, the N,N-[14C]dicyclohexylcarbodiimide-binding 16-kDa polypeptide and a 13-kDa polypeptide that also reacted with anti-ATPase and copurified with the tonoplast ATPase activity during gel filtration remained in the pellets after the wash treatments. We conclude that the 72- and 60-kDa polypeptides appear to be peripheral subunits of the tonoplast ATPase and that the 16-kDa polypeptide is probably embedded in the membrane bilayer. Additional subunits of the ATPase complex may include a 41-kDa (peripheral) and a 13-kDa (integral) polypeptide. Based on these results, a working model of the tonoplast ATPase analogous to the F1F0-ATPase is proposed.     

Fetal-maternal transfer of [H3]-6 beta-hydroxycortisol in the pregnant ewe

Distribution and fetomaternal transfer of 6 beta-hydroxycortisol (6 beta-OHF) was studied using serial sampling following injection of tritium labelled 6 beta-OHF into various fluid compartments in the chronically cannulated unaesthesized pregnant ewe. There was a rapid transfer of 6 beta-OHF from the fetal circulation into amniotic fluid and maternal blood. In contrast, the maternal----fetal transfer of this steroid metabolite was considerably less. The sequence of appearance of 6 beta-OHF in fetal blood and amniotic fluid following injection into maternal blood suggests that this steroid is first transferred across the placenta to fetal blood before gaining entry into the amniotic fluid space. The half-lives of 6 beta-OHF after initial equilibration in maternal plasma, fetal plasma and amniotic fluid were 2.0 h, 5.1 h and 8.9 h respectively. The amniotic sac appears to contain a relatively static pool of 6 beta-OHF and may act as a "trap" for 6 beta-OHF in the ovine conceptus.     

Immunohistochemical localization of two angiotensin I-converting isoenzymes in the reproductive tract of the male rabbit

The male reproductive tract contains two different isoenzymes of angiotensin I-converting enzyme (ACE), i.e., pulmonary and testicular ACE. The present study shows selectively the cellular distribution of the ACE isoenzymes in the reproductive tract of male rabbit, using indirect immunofluorescence or immunoperoxidase methods. Testicular ACE was found in the seminiferous tubules of the testes in spermatocytes containing mature spermatids, and in spermatids within the epididymal tubular lumen in sexually mature, but not in immature, rabbits. Epididymal tubular cells contained pulmonary ACE. In the young rabbit, epididymal tissue contained more ACE than that in adult rabbit, since ACE was observed in principal cells in addition to basal cells. In mature rabbit, ACE was observed in basal cells only. Strong staining for pulmonary ACE was observed in cells of the vas deferens in both young and adult rabbit. Therefore, synthesis of epididymal ACE, unlike the testicular isoenzyme, was not stimulated by sexual maturation. Enzymatically active ACE in seminal fluid corresponds to the pulmonary isoenzyme. The present study indicates that this seminal fluid ACE may originate from cells of the epididymal tubules, particularly those of the vas deferens. Endothelial cells of blood vessels lying in the interstitium of both testicular and epididymal tissue contained the pulmonary isoenzyme.     

Modulation of postinhibitory increase in plasma prolactin by domperidone in patients with prolactin secreting adenoma

To evaluate the PRL secretory mechanism in patients with PRL-secreting adenoma (PRL-oma), plasma PRL responses to dopamine (DA) were studied in these cases and in normal subjects. Plasma PRL values showed clear decreases during the infusion of DA (5 micrograms/kg/min for 90 min) in both 6 normal and 7 PRL-oma subjects (%decrease: 43.8 +/- 3.9% vs. 53.9 +/- 5.6%; NS) and postinhibitory increases after the termination. However, the postinhibitory increase occurred more promptly and markedly in PRL-oma patients than in normal subjects, i.e. the postinhibitory increase exceeded the basal level 45 min after the termination of DA infusion in PRL-oma patients, whereas the increase in normal subjects did not exceed the basal level even 90 min after the infusion. When domperidone was injected at the termination of DA infusion, the postinhibitory increases were significantly enhanced in either PRL-oma or normal subjects. The maximal increments in plasma PRL in the combination test of DA plus domperidone were significantly larger in PRL-oma patients, but were almost the same in normal controls, compared to the single domperidone test. In contrast, TRH did not modify the postinhibitory rises in 9 PRL-oma patients. These results indicate that the secretory properties and the sensitivities of lactotrophs to decreasing action of DA might be different between PRL-oma patients and normal controls. Further, the postinhibitory rebound phenomenon in PRL-oma patients is possibly determined by an overshoot of PRL storage concomitantly with a decreasing DA action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+/calmodulin-dependent protein kinase II. Isozymic forms from rat forebrain and cerebellum

Ca2+/calmodulin-dependent protein kinase II, an abundant brain protein proposed to mediate a number of Ca2+-regulated processes in neuronal tissue, is composed of autophosphorylatable subunits of Mr 50,000 and 60,000/58,000. A recent study (McGuinness, T. L., Lai, Y., Greengard, P., Woodgett, J.R., and Cohen, P. (1983) FEBS Lett. 163, 329-334) suggested that this kinase exists as isozymes which vary in the relative ratio of these subunits in different tissues or species. Other studies (Walaas, S. I., Nairn, A. C., and Greengard, P. (1983) J. Neurosci. 3, 291-301, 302-311) provided evidence which suggested that the ratio of these phosphopeptides might vary in different brain regions. In the present investigation, we have tested this possibility by comparing Ca2+/calmodulin-dependent protein kinase II purified from rat forebrain and cerebellum. The two kinases had similar purification characteristics, subunit compositions, physical properties, and substrate specificities. Gel filtration and sucrose density gradient centrifugation provided an estimated molecular weight of 550,000 for the forebrain kinase and 615,000 for the cerebellar kinase. The kinases from the two regions clearly differed in the relative proportions of the Mr 50,000 and 60,000/58,000 subunits. Three independent methods indicated that the forebrain kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 3:1 ratio, while the cerebellar kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 1:4 ratio. The forebrain kinase subunits were shown to be identical to the corresponding subunits of the cerebellar kinase by several criteria. The data are consistent with the existence in various brain regions of isozymic forms of Ca2+/calmodulin-dependent protein kinase II which differ in their relative subunit ratios.     

Specific absorption rate in rats exposed to 2,450-MHz microwaves under seven exposure conditions

Both positive and negative biological effects of microwaves on drug actions in rats exposed to 1-mW/cm2, 2,450-MHz microwaves have been reported by several investigators. We conducted dosimetry studies for seven different exposure conditions to determine whether these different results could be due to the rats having been exposed differently. They included anterior and posterior exposures in a circular waveguide, near field, far field with E- or H-field parallel to the long axis of the body and dorsal exposure in a miniature anechoic chamber with E- or H-field parallel to the long axis of the body. The average specific absorption rates (SARs) in the head, tail, and body of the exposed rats were measured by means of a calorimetry system. The local SARs at eight locations in the brain were determined by temperature measurement with Vitek probes. Intensive coupling of energy to the tail when it was exposed parallel to the E-field was shown by thermography. For the same average incident power density, the average SARs in the heads of rats were about two times higher in the circular waveguide than for other exposures. The local SARs in the brain varied for different exposure conditions. Statistical comparisons of SARs under the different exposure conditions are presented.