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Cholinesterases in aquatic biomonitoring: assay optimization and species-specific characterization for a California native fish.

Cholinesterase (ChE) enzyme activity measurements are widely applied in aquatic organisms for water quality monitoring, especially for pesticide contamination in agricultural watersheds. These biomarkers are amenable to measurement in a variety of species, and are therefore useful for examining effects in model organisms relevant to the ecosystem of interest. However, extensive variation in ChE biochemistry exists among tissues and species. This variation is rarely characterized and may lead to biases in the interpretation of activity determinations. We optimized ChE activity measurement parameters and characterized ChE biochemistry in Sacramento sucker (Catostomus occidentalis), a widely distributed fish native to watersheds of the Central Valley of California. Acetylcholinesterase (AChE) was the predominant ChE present in C. occidentalis brain and muscle, and muscle AChE was most sensitive to diazinon inhibition. Field caging experiments indicated that exposures to ChE-inhibiting pesticides were insufficient to induce neurotoxic effects. However, pesticide usage in the Central Valley is highly variable among years, and long-term monitoring of in-stream effects would be necessary to evaluate trends in pesticide contamination. Recent changes to the State Water Code require agricultural landowners to participate in a regional water quality monitoring plan. As with most regional monitoring plans, measurements of in-stream effects, and effects in resident species, are not scheduled to be included. We suggest that inclusion of biomarker measures would lend important information to the monitoring process, and propose these procedures as a template for adapting ChE activity measurements into region-specific monitoring programs to assess in-stream effects of pesticide contamination on native species.

Acetylcholinesterase↗

Primary structure of the serotonin transporter in unipolar depression and bipolar disorder.

Genetic factors have been implicated in the etiology of affective disorders but due to the complex inheritance patterns of these disorders, identification of the responsible gene(s) has so far been unsuccessful. Decreased platelet serotonin (5-HT) transport and reduced binding of imipramine or paroxetine to brain and platelet 5-HT uptake sites/transporters in patients with depression and suicide victims define the 5-HT transporter (5-HTT) as a candidate gene. The primary structure of the 5-HTT was analyzed in 17 patients meeting DSM-III-R diagnostic criteria for major depressive or bipolar disorder and in 4 healthy controls using polymerase chain reaction (PCR-) amplification and sequencing of complementary deoxyribose nucleic acid (cDNA) synthesized from platelet 5-HTT messenger ribose nucleic acid (mRNA). Direct PCR sequencing of the protein coding region failed to reveal changes in the deduced amino acid sequence of the platelet/brain 5-HTT (40,000 base pairs sequence screened), although a conservative single-base substitution representing a silent polymorphism was found. The results provide preliminary evidence that alterations in the primary structure of 5-HTT are not generally involved in the pathogenesis of unipolar depression and manic-depressive illness.

Adult↗

Up-regulation and down-regulation of cell surface and mRNA expression of CD5 antigen by various humoral factors on murine 70Z/3 pre-B cell leukemia cell line: IL-4 down-regulates CD5 antigen expression.

CD5, a pan-T cell antigen, is expressed on a minor subset of normal B lymphocytes and on cells of most B lineage tumors or transformed B cells in both man and animal models. In the present study, the effects of various humoral factors on CD5 expression by cells of a subcloned 70Z/3 murine pre-B leukemia cell line were investigated. Among the humoral factors studied, only LPS up-regulated CD5 expression on 70Z/3 cells (three- to fourfold) in a dose-dependent manner. However, this up-regulatory effect of LPS was not observed when cells were cultured in serum-free medium. NZB-serum factor (NZB-SF), a cytokine we have identified and shown to enhance the maturation and proliferation of immature B cells, synergistically enhanced CD5 expression in the presence of suboptimal doses of LPS. IL-4 down-regulated CD5 expression by 70Z/3 cells induced by LPS or LPS plus NZB-SF in a dose-dependent manner. IL-4 also suppressed spontaneous CD5 expression by 70Z/3 cells. No other cytokine tested showed an inhibitory effect. LPS, IFN-gamma, NZB-SF, and IL-1 enhanced sIg expression on 70Z/3 cells and their action on sIg expression was not inhibited by IL-4. Thus, the down-regulatory action of IL-4 on CD5 expression appeared specific for this antigen. IFN-gamma, which inhibits IL-4 induced CD23 and DR expression on B cells, does not abolish the down-regulatory action of IL-4 on CD5 expression by 70Z/3 cells. Changes in mRNA levels on coding CD5 were also examined following the incubation of 70Z/3 cells (24 hr) in the presence of humoral factors which can influence CD5 Ag expression. The levels of mRNA for CD5 Ag were moderately increased in the presence of LPS and NZB-SF. IL-4 appeared to suppress the actions of NZB-SF and LPS at least in part by reducing the levels of mRNA encoding CD5.

Animals↗

Neuroendocrine rhythms.

Hormones are secreted with circhoral, circadian and seasonal periodicities. Circhoral pulsatility is a temporal code, many chronic and acute changes in neuroendocrine status being mediated by changes in the frequency of circhoral release. The identity of the neuronal circuits controlling circhoral release is not known. Circadian release of hormones occurs with a precise temporal order entrained to the light-dark cycle, synchronized to the activity/rest rhythm and generated by circadian oscillators, of which the suprachiasmatic nuclei are the most important. Seasonal rhythms are driven either by an endogenous circannual clock mechanism or by a process of photoperiodic time measurement which is dependent upon the duration of the nocturnal peak of the pineal hormone melatonin.

Animals↗

The preexcitation syndrome: epidemiological and genetic study.

A sample of 4210 subjects of both sexes aged 35-54 years was examined, chosen at random from six regions of Croatia. An electrocardiogram at rest was performed in all subjects and changes analyzed by the Minnesota code. A short P-R interval together with a widening QRS complex and a delta wave was found in 0.05%, while 42 (1.0%) of the examinees had a short P-R interval, but only 0.21% were symptomatic. Three years after the first examination 0.06% of the subjects had preexcitation with a delta wave, and in one subject it appeared after three years. 0.35% of the subjects had a short P-R interval after three years but only 0.18% were symptomatic and in 22 (0.65%) it had disappeared in three years. After 13 years these subjects did not appear for an examination, and the short P-R interval did not appear in any of other subjects during this period. There were more short P-R intervals: 3.22% in females and 1.96% in males, but 0.33% only were symptomatic. Antigens of the human leukocyte group A (HLA) system were analyzed in 46 patients: the Wolff-Parkinson-White syndrome was found in 35, while 11 had the Lown-Ganong-Levine syndrome. Antigens of the HLA-A, HLA-B and HLA-DR locuses were determined by the microlymphocytotoxicity method. The results of the frequency of HLA system antigens were compared to the results of the control group of a Croatian population consisting of 175 people. There was an increased frequency of HLA-A9 and HLA-B5 (P = 0.026 and 0.0092) in the investigated population as a whole. The participation of HLA-A3 antigen was significantly less among patients (P = 0.03), while HLA-B14 antigen was not found in patients with preexcitation. Within 10 HLA-DR locuses, HLA-DR7 antigen was rather more frequently present, although this was not statistically significant (P = 0.173).

Adult↗

Tryptophan hydroxylase inhibition increases preprotachykinin mRNA in developing and adult medullary raphe nuclei.

In order to study the regulation of co-localized monoamine and peptide neurotransmitters in the medullary raphe nuclei (MRN), we determined whether inhibition of serotonin (5-HT) synthesis affected levels of preprotachykinin (PPT; the prohormone precursor of substance P) mRNA in the MRN. Adult rats received p-chlorophenylalanine (pCPA), an irreversible inhibitor of tryptophan hydroxylase (TPH), via Alzet minipumps. TPH activity was inhibited by 70-80% for 3 weeks following pump implantation. During this period Northern mRNA analysis revealed that PPT mRNA levels in the MRN were increased 1.5-2-fold. The pCPA-induced increase was specific for PPT mRNA since no change was detected in mRNA coding for neuron-specific enolase (NSE; a constitutive neuronal protein) or 28 S ribosomal RNA. To determine whether fetal inhibition of 5-HT synthesis affected development of PPT mRNA in the MRN, pregnant rats were administered pCPA via Alzet minipump implanted on embryonic day 8. In pCPA-treated litters TPH activity was decreased by 60-70% from E16 to postnatal day 3 (P3), returning to control levels by P8. Northern mRNA analysis revealed that PPT mRNA levels increased 2.4-fold of control levels at P1. Infusion of pCPA for one week resulted in an earlier increase in PPT mRNA levels, suggesting that birth was not required to elicit the surge in PPT message. These results support the hypothesis that alterations in 5-HT metabolism have regulatory consequences for co-localized substance P formation in the MRN.

Animals↗

GABAA and glutamate receptor subunit mRNAs in cortex of mice chemically kindled with FG 7142.

Messenger RNA (mRNA) for several subunits of the GABAA receptor was measured in the cortex of mice chemically kindled with FG 7142. At 10 days after the final FG 7142 injection, beta 2 and gamma 2S subunit mRNA were significantly increased. At 31 days, alpha 1, alpha 3, beta 2, and gamma 2L mRNA were elevated. In contrast, levels of mRNA for four subunits of the glutamate receptor in the cortex of FG 7142-kindled mice killed at 31 days were not significantly increased. Previous investigations have shown a reduction in GABA-gated chloride channel function and density in mice kindled with FG 7142, and the increases in subunit mRNA found in the present studies may be a response to these decreases. These results indicate that chemical kindling produces long-lasting changes in expression of genes coding for specific neurotransmitter receptor subunits.

Animals↗

Isolation of a rat S100 alpha cDNA and distribution of its mRNA in rat tissues.

In order to clarify the reported discrepancies in S100 alpha protein and mRNA distribution in rat tissues, a rat S100 alpha cDNA has been isolated and this species homologous probe along with a rat S100 beta cDNA probe has been used to examine S100 mRNA expression in rat tissues. Although the rat S100 alpha cDNA was missing approximately 30 nucleotides of coding sequence, only 4 conservative changes in amino acid sequence were observed when the deduced amino acid sequence was compared to the bovine S100 alpha amino acid sequence. Thus, S100 alpha proteins, like S100 beta proteins, are highly conserved among species. All nineteen of the tissues examined (including cerebrum and cerebellum) contained S100 alpha mRNA. In addition, S100 beta mRNA was detected in thirteen of the nineteen tissues examined. These results are in agreement with previous protein distribution studies and further demonstrate that S100 proteins are not brain-specific and are expressed in a large number of tissues. Although S100 alpha and S100 beta mRNAs were detected in rat tissues which had previously been reported to contain S100 alpha and S100 beta protein, a direct correlation between the protein and mRNA levels were not observed, suggesting that different mechanisms regulate S100 expression in various tissues. S100 alpha exhibited a single similar size mRNA species (0.5 Kb) in all tissues examined, as did S100 beta (1.5 Kb), suggesting that the individual S100 proteins are expressed as single mRNA and protein products in rat tissues.

Amino Acid Sequence↗

Selection for the expression of one form of Chinese hamster dihydrofolate reductase over another during growth in methotrexate.

Two mammalian expression plasmids, each carrying a cDNA encoding a different allele of dihydrofolate reductase (DHFR) present in the Chinese hamster lung cell line DC-3F, were constructed. These simian virus 40 promoter-enhancer-based plasmids, designated pSVA75 and pSVMQ19, are identical except for a G-->A transition at nucleotide 286 of the DHFR coding sequence. Due to this change, the enzyme expressed by pSVA75 contains Asp95, while the enzyme expressed by pSVMQ19 has Asn95 [Melera et al., J. Biol. Chem. (1988) 1978-1990]. Both forms of the enzyme are catalytically equivalent and are produced to similar levels in DC-3F cells [Yu and Melera, Cancer Res. (1993) 6031-6035; H.Y., A.H. and P.W.M., in preparation]. Clonal cell lines expressing one or the other DHFR allele were obtained via transfection of DHFR- Chinese hamster ovary cells, and 74 clones of each type isolated. These were pooled and divided into 40 aliquots, each of which was then subjected to selection by growth in sequentially increasing concentrations of methotrexate (MTX). Analysis of the resulting drug-resistant populations revealed that cells producing Asp95 DHFR dominated with an overall frequency of 3:1, and therefore, under these growth conditions, display a selective advantage over those producing Asn95 DHFR. These data extend previous observations showing that independent selections of the heterozygous parent cell line DC-3F in MTX result in threefold more MTX-resistant lines overexpressing the Asp95-encoding DHFR allele than the Asn95-encoding DHFR allele.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

Characterization of the human cDNA and genomic DNA encoding CART: a cocaine- and amphetamine-regulated transcript.

PCR differential display screening has recently identified a rat mRNA termed CART (cocaine- and amphetamine-regulated transcript) which is transcriptionally regulated in the striatum following acute administration of psychomotor stimulants. The endogenous CART transcript is expressed in diverse rat brain structures, as well as endocrine tissues. The deduced CART protein contains a hydrophobic signal sequence, suggesting that it may be targeted for secretion. Thus, the CART protein may represent a novel neuroendocrine signaling molecule. The study described here represents a complete analysis of the human CART cDNA and gene. The complete nucleotide (nt) sequence of the approx. 900-nt human CART transcript is contained within three distinct exons, with the entire human CART gene localized to a segment of genomic DNA approx. 2 kb in length. The human CART cDNA sequence is 80% identical to the corresponding rat cDNA, with 92% homology observed within the deduced protein-coding region. Third-nt changes account for most of the latter differences, with CART exhibiting 95% identity between these two species at the amino-acid sequence level. PCR/Southern blot analysis of DNA isolated from human/rodent somatic cell hybrid panels localizes the CART gene to human chromosome 5. Lastly, Northern blot analysis reveals that the gross pattern of distribution of CART mRNA in human brain is similar to that previously observed in rat. These overall similarities suggest that CART plays a conserved role within the mammalian neuroendocrine system.

Amino Acid Sequence↗

Differential effect of dietary spermine on alkaline phosphatase activity in jejunum and ileum of unweaned rats.

Spermine is a low molecular weight polyamine involved in the postnatal maturation of the gut. When it is administered orally to suckling rats it induces the maturation of their spleen, liver, pancreas, and small intestine. We showed that this polyamine modulates differently the activity of alkaline phosphatase in jejunum and ileum in suckling rat. In 14-day-old rat which had received spermine orally for 3 days, once daily, an increase of alkaline phosphatase activity in the jejunum and a decrease of this activity in the ileum was observed. Alkaline phosphatase was located at the bottom of the villus in the control jejunum and in the whole length of the villus in spermine-treated rats. On the contrary, in ileum of controls, this enzyme was present in the whole length of the villus but disappeared in the spermine-treated animals. An enzyme mass shift was observed in the small intestine after spermine administration. Spermine administration did not change the expression of genes coding for alkaline phosphatase, suggesting a post-transcriptional modification.

Administration, Oral↗

The analysis of interaural time differences in the chick brain stem.

The brain stem auditory system of the chick has proven to be a useful model system for analyzing how the brain encodes temporal information. This paper reviews some of the work on a circuit in the brain stem that compares the timing of information coming from the two ears to determine the location of a sound source. The contralateral projection from the cochlear nucleus, nucleus magnocellularis (NM), to nucleus laminaris (NL) forms a delay line as it proceeds from medial to lateral across NL. NL neurons function like coincidence detectors in that they respond maximally when input from the two ears arrive simultaneously. This arrangement may allow NL to code sound space by the relative level of activity across the nucleus. The head anatomy of the chick allows for enhancement of the functional interaural time differences. Comparing the functional interaural time differences to the length of the neural delay line suggests that each NL can encode approximately one hemifield of sound space. Finally it is suggested that inhibitory input into the NM-NL circuit may provide a means to dynamically adjust the gain of the circuit to allow accurate coding of sound location despite changes in overall sound intensity.

Acoustic Stimulation↗

Extensive induction of important mediators of fibrosis and dystrophic calcification in desmin-deficient cardiomyopathy.

Mice lacking the intermediate filament protein desmin demonstrate abnormal mitochondria behavior, disruption of muscle architecture, and myocardial degeneration with extensive calcium deposits and fibrosis. These abnormalities are associated with cardiomyocyte hypertrophy, cardiac chamber dilation and eventually with heart failure. In an effort to elucidate the molecular mechanisms leading to the observed pathogenesis, we have analyzed gene expression changes in cardiac tissue using differential display polymerase chain reaction and cDNA atlas array methods. The most substantial changes were found in genes coding the small extracellular matrix proteins osteopontin and decorin that are dramatically induced in the desmin-null myocardium. We further analyzed their expression pattern both at the RNA and protein levels and we compared their spatial expression with the onset of calcification. Extensive osteopontin localization is observed by immunohistochemistry in the desmin-null myocardium in areas with massive myocyte death, as well as in hypercellular regions with variable degrees of calcification and fibrosis. Osteopontin is consistently co-localized with calcified deposits, which progressively are transformed to psammoma bodies surrounded by decorin, especially in the right ventricle. These data together with the observed up-regulation of transforming growth factor-beta1 and angiotensin-converting enzyme, could explain the extensive fibrosis and dystrophic calcification observed in the heart of desmin-null mice, potentially crucial events leading to heart failure.

Animals↗

Increased myocardial expression of RAMP1 and RAMP3 in rats with chronic heart failure.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are potent vasodilators in humans and improved myocardial ischemia is observed after CGRP administration. Receptors for CGRP and ADM were already identified in heart. Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a CGRP receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an ADM receptor. As CGRP and ADM may play a beneficial role in heart failure, we investigated whether the CGRP and ADM receptors are upregulated in chronic heart failure. We have used semi-quantitative RT-PCR and Western-blot analysis to detect and quantify the mRNA and the protein of RAMP1 and RAMP3 in both atria and ventricles of failing hearts 6 months after aortic banding in rats. Our results showed for the first time an up-regulation of RAMP1 and RAMP3 mRNAs and proteins in this model of cardiac failure. No change was observed in mRNAs coding for CRLR, RAMP2, RDC1 (canine orphan receptor), and ADM. The present results suggested after congestive heart failure in adult rats, an up-regulation of the CGRP receptor (by an increase in RAMP1 that is associated with CRLR) in atria and ventricles and of ADM receptor (by increased RAMP3 expression that is associated with CRLR) in atria. These findings support a functional role for CGRP and ADM receptors to compensate the chronic heart failure in rats.

Adrenomedullin↗

Transcript mutations of the alpha regulatory subunit of protein kinase A and up-regulation of the RNA-editing gene transcript in lupus T lymphocytes.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder characterised by diverse dysfunctions of immune effector cells, including proliferation and cytotoxicity. In T cells from patients with SLE, activity of type 1 protein kinase A isozymes is greatly reduced because of decreased expression of the alpha and beta regulatory subunits (RI alpha and RI beta). We aimed to identify a molecular mechanism or mechanisms for this isozyme deficiency by assessing occurrence of mutations in transcripts of the RI alpha subunit in patients with SLE. METHODS: We cloned and sequenced cDNA of RI alpha and corresponding genomic DNA of the coding region to detect sequence changes from eight patients with SLE and six healthy controls. Because transcript editing is regulated by adenosine deaminases that act on RNA (ADAR), we quantified expression of ADAR1 transcripts in SLE and control T cells by competitive PCR. FINDINGS: Sequence analyses of cDNA showed heterogeneous transcript mutations, including deletions, transitions, and transversions. We identified 1.22 x 10(-3)/bp transcript mutations in SLE T cells-a frequency 7.5 times higher than that in control T cells. By contrast, we identified no genomic mutations. Two hotspots were identified in the RI alpha subunit transcripts from SLE T cells, one located adjacent to a pseudosubstrate site of the RI alpha subunit and the other a component of the cAMP binding A domain. ADAR1 mRNA content was 3.5 times higher in SLE cells than in control T cells (p=0.001). INTERPRETATION: An RNA-editing enzyme could be converting adenosine to inosine within double-stranded regions of RNA, resulting in transcript mutations. This process could be one mechanism resulting in mutations in the RI alpha subunit of type 1 protein kinase A.

Adenosine Deaminase↗

Force of limb withdrawals elicited by graded noxious heat compared with other behavioral measures of carrageenan-induced hyperalgesia and allodynia.

We investigated if carrageenan-induced thermal hyperalgesia and mechanical allodynia are associated with a reduction in threshold and-or enhancement of suprathreshold nocifensive responses, using a method to measure the force of a hind limb wilhdrawal reflex elicited by graded noxious heat stimuli (36-52 degrees C, 5s) delivered by Peltier thermode tethered to the ventral hind paw of conscious rats. Withdrawal reflexes were recorded 2.5 h after intraplantar injection of carrageenan (1 or 0.1%) or saline vehicle in sessions >2 weeks apart: baseline reflexes were assessed the day before. Withdrawal reflex force increased linearly from 42-52 degrees C. Carrageenan 1% significantly enhanced withdrawal reflexes at 40-46 degrees C, reducing the slope and threshold of the stimulus-response function. This was associated with significant reductions in thermal paw withdrawal latency (Hargreaves test: by 50%), mechanical withdrawal threshold (by 82%) and weight bearing on the injected side (by 81%) measured with independent force plates. Smaller reductions in thermal paw withdrawal latency and mechanical withdrawal threshold, and smaller enhancement of withdrawal reflex force, were observed following 0.1% carrageenan. Intraplantar saline was ineffective. This method allows assessment of hyperalgesic changes in stimulus-response coding over a broad range of noxious stimulus intensities.

Animals↗

Evaluation of ribosomal RNA and actin gene sequences for the identification of ascomycetous yeasts.

Highly similar gene sequences of the 5' region of the large subunit (LSU) are commonly interpreted to predict the organism's identity. However, it was recognised that closely related taxa do not always show sufficiently diverged D1/D2 LSU sequences to differentiate between them. The effectiveness of species separation using D1/D2 LSU sequences, small subunit (SSU) sequences and actin gene sequences was determined by pair-wise comparisons. The LSU data showed coinciding similarities among and within species. The actin data resolved all investigated species. Examples strengthened the value of almost complete SSU sequences for species separation. The larger number of differences in the highly conserved actin gene, compared to the overall more variable LSU gene, is due to the tolerance of protein coding genes to synonymous nucleotide changes. In contrast, the pairing in secondary structures of the rRNA, ensuring the functionality of the molecule, relies on longer and uninterrupted sequence sections. In conclusion, D1/D2 LSU sequences are not specific enough to identify closely related taxa. The actin gene is a better marker in these cases. However, because of the availability of a large database of fungal D1/D2 LSU sequences, this gene region is currently still the preferred target for sequence-based identification.

Actins↗

A six-year retrospective analysis of shoulder dystocia and delivery of the shoulders.

OBJECTIVE: To investigate the diagnosis, incidence and management of shoulder dystocia. DESIGN: Retrospective review of all the deliveries between 1988 and 1993 which were coded on the computer data base as 'moderate difficulty with the shoulders' or 'shoulder dystocia'. SETTING: A teaching hospital in the south of England. PARTICIPANTS: The study population consisted of 15,658 women who had cephalic vaginal deliveries of babies weighing > 2.5 kg. The sample consisted of the 257 women reported to have moderate difficulty with the shoulders or shoulder dystocia at delivery. FINDINGS: There was a significant fall in the reported incidence of shoulder dystocia during the period under investigation. Eighty-six (1.1%) of the deliveries between 1988 and 1990 were reported to have been complicated by shoulder dystocia. In 1991 a second option was introduced to allow both shoulder dystocia or moderate difficulties with the shoulders to be coded after delivery. Following this change in categorisation, the incidence of shoulder dystocia was reduced to 30 (0.6%) reported cases in 1992-1993. Over the same period the reported incidence of moderate difficulty rose significantly from 29 (1.1%) in 1992 to 60 (2.4%) in 1993 (P < 0.001). Disparity was noted between the experience of practitioners and contemporary definitions of shoulder dystocia and this was highlighted by the number of reported cases of moderate difficulty. In this study practitioners used the term shoulder dystocia in a general sense to describe a range of difficulties encountered with the delivery of the shoulders and they identified many varied manifestations. In comparison, contemporary literature describes shoulder dystocia as a discrete entity. KEY CONCLUSIONS: Shoulder dystocia is a complex clinical scenario and perceptions of the incidence may be influenced by alterations in the delivery technique and changes in the diagnosis and documentation. IMPLICATIONS FOR PRACTICE: The application of traction may interfere with the normal mechanisms of labour and the spontaneous birth of the shoulders, whilst increasing the risk of trauma to the baby. It may be unnecessary to use such interventions routinely in the care of normal labouring women.

Birth Weight↗