Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Cloning, sequencing and distribution of the Salmonella typhimurlum LT2 siaiidase gene, nanH, provides evidence for interspecies gene transfer

The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli. Sialidase expression was regulated positively by cAMP. In contrast, certain Tn1000 insertions located upstream of nanH coding sequences reduced sialidase activity. A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy in S. typhimurium LT2. The complete nucleotide sequence of nanH, encoding a 41,300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragilis, and Trypanosoma cruzi. Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S. typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event. At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases. The predicted secondary structure of the bacterial enzymes was strikingly similar to viral sialidase. From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S. typhimurium obtained its nanH copy most recently from Salmonella arizonae. S. typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH. These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.     

Cytochrome P450s in the guinea pig adrenal that are immunologically similar to liver forms: estrogen suppression explains male-female differences

Several cytochrome P450s have been identified in guinea pig adrenal microsomes which are distinct from the known steroidogenic P450s, c17 and c21, and are immunochemically related to cytochrome P450s found in liver. One, a 52 K protein related to P450 I (CYP1), occurs almost exclusively in males, is localized to the inner zone, and is suppressed by ACTH. Its levels correlate with microsomal capacity for xenobiotic metabolism. The others, related to P450s II and III (CYP2 and 3), are more predominant in males, but not exclusive to them, are found in both the inner and outer zones, and are not suppressed by ACTH. Their functions remain to be elucidated. The male predominance of the CYP1-related protein has recently been shown to be due to suppression of the protein in females by estrogen. To determine if estrogen is also involved in the regulation of the CYP2-related proteins, ovariectomized and sham-operated animals were treated with a long-acting estrogen, estradiol valerate, or with the vehicle alone. These P450s reached male levels in ovariectomized females treated only with the vehicle. Their enhanced levels were suppressed by treatment with estrogen. Estrogen treatment also suppressed the levels of the P450s seen in sham-operated females. Endogenous estrogen produced similar effects. In hemi-ovariectomized females the contralateral ovary hypertrophied, a state in which estrogen levels would be maintained or increased. In these females no increase occurred in the immunodetectable P450s. In normal females, estrogen levels are low in prepubertal animals, rise at the time of puberty and drop again after ovarian cycling is completed. The CYP2-related proteins were present in adrenal microsomes of prepubertal females, but were suppressed after puberty. On the other hand, post-estrous females, in whom estrogen levels would be low, acquired male levels of these proteins in their adrenal microsomes. P450c17 and P450c21, as well as 3β-hydroxysteroid dehydrogenase, were not affected by surgery or estrogen. Taken together, these experiments indicate that suppression by estrogen in females can account, in large part, for the predominance of several immunochemical homologs of liver P450s in adult male guinea pig adrenals.     

Transient presence and functional interaction of endogenous GABA and GABAA receptors in developing rat optic nerve.

GABA (gamma-aminobutyric acid) is a major inhibitory synaptic neurotransmitter with widespread distribution in the central nervous system (CNS). GABA can also modulate axonal excitability by activation of GABAA receptors in CNS white matter regions where synapses and neuronal cell bodies are not present. Studies on cultured glia cells have revealed the synthesis of GABA in rat optic nerve O-2A progenitor cells that give rise to oligodendrocytes and type 2 astrocytes in vitro. We report here that: (i) GABA is detected by immuno-electron microscopy in intact rat optic nerve and is localized to glia and pre-myelinated axons during the first few weeks of postnatal development, but is markedly reduced or absent in the adult; and (ii) neonatal optic nerve is depolarized by GABAA receptor agonists or by the inhibition of GABA uptake. These results demonstrate the presence of functional GABAA receptors, and GABA uptake and release mechanisms in developing rat optic nerve, and suggest that excitability of developing axons can be modulated by endogenous neurotransmitter at non-synaptic sites.     

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.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.     

Cofactor interactions and the regulation of glutamate decarboxylase activity

More than 50% of glutamate decarboxylase (GAD) in brain is present as apoenzyme. Recent work has opened the possibility that apoGAD can be studied in brain by labeling with radioactive cofactor. Such studies would be aided by a compound that inhibits specific binding. One possibility is 4-deoxy-pyridoxine 5-phosphate, a close structural analog of the cofactor pyridoxal 5-phosphate. The effects of deoxypyridoxine-P on the cyclic series of reactions that interconverts apo- and holoGAD was investigated and found to be consistent with simple competitive inhibition of the activation of apoGAD by pyridoxal-P. As expected from the cycle GAD was inactivated when incubated with glutamate and deoxypyridoxine-P even though cofactor was present, but no inactivation was observed with deoxypyridoxine-P in the absence of glutamate. Deoxypyridoxine-P also stabilized apoGAD against heat denaturation. These effects were quantitatively accounted for by a kinetic model of the apo-holoGAD cycle. Deoxypyridoxine-P inhibited the labeling by [³²P]pyridoxal-P of GAD isolated from rat brain. Hippocampal extracts were labeled with [³²P]pyridoxal-P and analyzed by SDS-polyacrylamide gel electrophoresis. Remarkably few bands were strongly labeled. The major labeled band (at 63 kDa) corresponded to one of the forms of GAD. Other strongly-labeled bands were observed at 65 kDa (corresponding to the higher molecular weight form of GAD) and at 69–72 kDa. Labeling of the 63- and 65-kDa bands was inhibited by deoxypyridoxine-P, but the 69–72 kDa bands were unaffected, suggesting that the latter were non-specifically labeled. The results suggest that the 63-kDa form of GAD makes up the majority of apoGAD in hippocampus.Special issue dedicated to Dr. Eugene Roberts.     

The malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to <6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism—Thr:Ala—at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148^thr) are designated Malmö A, and negative reactors (148^ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and “lyonization” is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over—including double crossing-over—appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies ～50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.