Lydia Villa-Komaroff

Transfection of PC12 cells with the human GAP-43 gene: effects on neurite outgrowth and regeneration

The neuronal growth associated protein GAP-43 is expressed at high levels during axonal growth and regeneration. In this report, we describe the transfection of the nerve growth factor (NGF)-responsive pheochromocytoma cell line PC12 with the human GAP-43 cDNA under the control of the Moloney murine leukemia virus long terminal repeat (MoMuLV LTR). Two PC12 subclones were isolated that constitutively expressed GAP-43 from the transfected cDNA and showed increased responsiveness to NGF. Of the two transfected PC12 subclones, the subclone expressing the most human GAP-43 RNA showed an accelerated initial neurite outgrowth response and a 10-fold increased sensitivity to NGF. Neurite regeneration was significantly enhanced in both transfected subclones and, in contrast to untreated PC12 cells, could occur transiently in the absence of added NGF. These results suggest that GAP-43 may potentiate the action of NGF on neurite initiation and regeneration.

Characterization of the Two Nonallelic Genes Encoding Mouse Preproinsulin

SummaryWe have cloned and sequenced the two mouse preproinsulin genes. The deduced amino acid sequences of the mature mouse insulins are identical to the published protein sequences. However, the nucleotide sequence indicates that the mouse I C-peptide has a deletion of two amino acids compared with the mouse II C-peptide. We used an S1 nuclease assay to confirm the presence of the deletion and to measure the ratio of transcripts from gene I to transcripts from gene II. The mouse preproinsulin I gene, like the rat gene I, is missing the second intervening sequence that normally interrupts the C-peptide region in other insulin genes. Comparison of the 5′ flanking sequences of the mouse and rat genes II indicates that they are homologous for at least 1000 base pairs. The preproinsulin I genes also share homology in their 5′ flanking DNAs; however, their homology to the preproinsulin II genes extends for only about 500 base pairs.

Changes in immediate early gene expression during postnatal development of cat cortex and cerebellum

Postnatal brain development involves interactions between extracellular signals and preprogrammed genetic events. Immediate early genes (IEGs) are a group of genes that are induced by extracellular signals and their protein products alter transcription by binding regulatory elements in other genes. Using Northern and slot blot analysis of total RNA isolated from visual cortex, frontal cortex, and cerebellum of cats, we have determined the postnatal development patterns of mRNA expression for 5 of these genes, c-fos, erg-1, c-jun, jun-B, and c-myc. Each gene had a distinct developmental pattern of mRNA expression, and for a given gene, these patterns were often different in different brain structures. These results suggest that temporal changes in the combinatorial interaction of different IEGs during early postnatal life are important for normal brain development.

Pancreatic Reg/pancreatic stone protein (PSP) gene expression does not correlate with beta-cell growth and regeneration in rats

SummaryThe Reg/pancreatic stone protein (PSP) gene is postulated to be an important regulator of pancreatic beta-cell growth. To investigate this hypothesis, we analysed the expression of the Reg/PSP gene following a 90% pancreatectomy and after chronic glucose infusion, two well-defined models of pancreatic beta-cell growth. There was a rapid induction of the Reg/PSP gene in the remnant pancreas after a 90% pancreatectomy in rats during the period of marked growth of the exocrine and islet tissue. However, a similar rapid, but smaller, induction of the Reg/PSP gene was observed in sham-operated rats and in non-surgical control rats in which there was no enhanced pancreatic growth. Furthermore, there was no pancreatic Reg/PSP gene induction in a model of selective beta-cell growth, the chronic glucose-infused rat. Thus, it is unlikely that Reg/PSP is a beta-cell specific growth factor, even though the function of this important pancreatic gene is still unknown.

Islet B-cell dysfunction and the time course of recovery following chronic overinsulinisation of normal rats.

SummaryAppropriate insulin therapy may preserve or improve islet B-cell function whereas the effects of overinsulinisation are unclear. Pancreatic islet B-cell function was therefore studied after overinsulinisation of normal rats for 4 weeks (fed blood glucose 2.2–4.5 mmol/l, controls 4.1–7.0 mmol/l). Insulin secretion was assessed by a 3-h hyperglycaemic clamp (10.0 mmol/l) performed 1, 48, and 120 h after insulin withdrawal (n=6 in each group). When the clamp was performed 1 h after insulin withdrawal, clamp insulin concentration was 1.6±0.1 μg/l, compared to 9.3±1.0 μg/l in control rats. The integrated area under the plasma insulin concentration curve was also significantly decreased (4.8±0.4 vs 20.3±2.2 μg·l−1·h−1, p<0.001), but recovered to 9.4±1.0 μg·l−1·h−1 after 48 h, and to 17.5±1.4 μg·l−1·h−1 after 120 h. Pancreatic insulin contents were decreased at 1 h (6±1 μg/g wet wt) and 48 h (54±12 μg/g wet wt) but not at 120 h (221±30 μg/g wet wt) after withdrawal (controls, 303±29 μ/g wet wt) and there was a strong relationship with pancreatic preproinsulin mRNA and the clamp insulin response. Thus, overinsulinisation with prolonged periods of low blood glucose concentrations impairs islet B-cell function, but is reversible over 5 days.

Expression of messenger ribonucleic acid for epidermal growth factor receptor and its ligands, epidermal growth factor and transforming growth factor-α, in human first- and second-trimester fetal ovary and uterus

OBJECTIVES The epidermal growth factor receptor, a 170 kd polypeptide with tyrosine kinase activity, is used as the common receptor by two homologous polypeptide growth factors, epidermal growth factor and transforming growth factor-alpha. The activation of the epidermal growth factor receptor results in effects including deoxyribonucleic acid synthesis and cellular differentiation. Epidermal growth factor, transforming growth factor-alpha, and the epidermal growth factor receptor are reported to be associated with adult reproduction. However, the pattern of gene expression for the epidermal growth factor receptor and its ligands in human fetal reproductive tissues has not been detailed previously. STUDY DESIGN We studied the expression of messenger ribonucleic acid encoding three polypeptides, epidermal growth factor receptor, epidermal growth factor, and transforming growth factor-alpha, in 10-, 15-, 19-, and 22-week human fetal ovaries and uteri. Ribonucleic acid was prepared from the fetal tissues and made into complementary deoxyribonucleic acid by reverse transcription. The complementary deoxyribonucleic acid was amplified by polymerase chain reaction, using primers specific for the human epidermal growth factor receptor, epidermal growth factor, and transforming growth factor-alpha. RESULTS We found that epidermal growth factor receptor messenger ribonucleic acid was present in all stages of ovarian and uterine tissues studied. In addition, both epidermal growth factor and transforming growth factor-alpha messenger ribonucleic acid was found in all four stages of ovarian development. Epidermal growth factor messenger ribonucleic acid was detected in all four stages of fetal uterine development. CONCLUSIONS This is the first demonstration, to our knowledge, of epidermal growth factor receptor, epidermal growth factor, and transforming growth factor-alpha messenger ribonucleic acid expression in human first- and second-trimester uterus and ovary.

The ratio of mouse insulin I:insulin II does not reflect that of the corresponding preproinsulin mRNAs

Rats and mice both express two, non-allelic, insulin genes. In the rat the ratio of the two preproinsulin mRNAs closely matches that of the mature insulin peptides. The experiments reported here demonstrate that this is not the case in the mouse. The relative amounts of the two murine proinsulin RNAs were measured by an S1 nuclease assay. The ratio of preproinsulin I mRNA to preproinsulin II mRNA was 4:1 in RNA extracted from the pancreas of mice fed ad libitum or fasted for 72 h. A similar value was found in mouse islets of Langerhans after maintenance in tissue culture for 48 h at either 2.8 or 16.7 mM glucose. The ratio of insulin I:insulin II peptides, assessed by separating the two insulins using reversed phase high-performance liquid chromatography, was approximately 1:3 in both pancreas and islets. Thus in the mouse, unlike the rat, the ratio of the two insulin peptides does not reflect that of the two preproinsulin mRNAs.

mRNAs for insulin-like growth factor-II (IGF-II) and variant IGF-II are co-expressed in human fetal ovary and uterus

Insulin-like growth factor-II (IGF-II) is postulated to have autocrine and/or paracrine functions in developing fetal tissues, but has never been reported in human fetal reproductive organs. The forms of IGF-II found in normal human serum include a 67 amino acid form and a variant form resulting from alternate splicing of the mRNA such that Ser-29 is replaced by four other amino acid residues. We studied the expression of mRNA encoding IGF-II in human fetal ovaries and uteruses of 10, 15, 19 and 22 weeks of gestation. By reverse transcription followed by polymerase chain reaction (PCR), we identified the co-expression of two mRNAs encoding IGF-II in all developmental stages of fetal ovaries and uteruses tested. One of the PCR amplified fragments was 9 nucleotides larger than the other. The PCR amplified ovarian and uterine DNA fragments were mapped by digestion with the restriction endonucleases AvaII and PvuII and both the IGF-II fragment and the larger IGF-II fragment produced the anticipated DNA patterns by gel electrophoresis. The PCR amplified DNA fragments were cloned and sequenced to confirm that the expressed mRNAs encoded IGF-II and variant IGF-II. We conclude that IGF-II and variant IGF-II mRNA co-expression occurs in the human fetal female genital tract and that the two forms of the growth factors may have physiologic roles in reproductive tract development.

Messenger ribonucleic acid for transforming growth factor-α, but not for epidermal growth factor, is expressed in fetal and neonatal mouse brain

OBJECTIVE Little is known concerning the presence of transforming growth factor-alpha in fetal and neonatal tissues. Our objective was to analyze messenger ribonucleic acid expression for transforming growth factor-alpha and epidermal growth factor, the structural and biochemical analog of transforming growth factor-alpha in fetal and neonatal murine brain. STUDY DESIGN Messenger ribonucleic acid was prepared from whole brains of mice from these developmental stages: embryonic days 14, 15, and 17, postnatal days 0, 4, 10, and adult. Polymerase chain reaction was performed on the complementary deoxyribonucleic acid obtained from reverse transcription of messenger ribonucleic acid with transforming growth factor-alpha and epidermal growth factor primers. In addition, ribonuclease protection assay was used to identify transforming growth factor-alpha transcripts. RESULTS We found messenger ribonucleic acid encoding transforming growth factor-alpha in all stages of development used. Epidermal growth factor messenger ribonucleic acid was not found in any stage. Ribonuclease protection assay confirmed transforming growth factor-alpha transcripts in these tissues. CONCLUSION Transforming growth factor-alpha may play an autocrine or paracrine role in the differentiation or maintenance of murine fetal and neonatal brains.

Expression and imprinting of the insulin-like growth factor II gene in neonatal mouse cerebellum

Insulin‐like growth factor II (IGF‐II) plays significant roles in the growth and development of mammals through the regulation of mitogenesis and cell survival. Previously, IGF‐II mRNA transcripts within the CNS were detected in the choroid plexus and leptomeninges (DeChiara et al., 1991). The objective of this study was to determine the expression pattern of IGF‐II mRNA in different cell types of the cerebellum during development. We report here that the IGF‐II gene is transcribed in granule and glial cells within the cerebellar parenchyma at various times during the early postnatal period in mice. IGF‐II gene expression is further regulated by parent‐specific imprinting such that only the paternal IGF‐II allele is expressed in granule cells. In contrast, choroid plexus and leptomeninges express IGF‐II mRNAs biallelically, indicating that cell type‐specific regulation of genomic imprinting occurs within the mammalian CNS. J. Neurosci. Res. 50:958–966, 1997. © 1997 Wiley‐Liss, Inc.