Molecular phylogenetic reconstruction improves the taxonomic understanding of Indian Dipcadi (Asparagaceae) and reveals a new species from the bank of Hiranyakeshi River, Maharashtra, India.

Dipcadi (Scilloideae: Asparagaceae) is a genus of bulbous monocots with approximately 40 species, of which 13 occur in India. Species delimitation within the genus has been troublesome hindering a comprehensive phylogenetic analysis. The most recent phylogeny of the subfamily Ornithogaloideae included six species of Dipcadi only from Africa. Here, we reconstructed the phylogeny of Ornithogaloideae including 23 accessions comprising 13 recognized taxa (11 species and two varieties) of Indian Dipcadi. The phylogenetic analyses were based on nucleotide sequences of three plastid regions (rbcL, matK and trnL-F spacer) and one nuclear region (ITS). Pseudogaltonia clavata exhibited sister relationship to Dipcadi. Our combined nuclear + plastid dataset analyses revealed a monophyletic Dipcadi with five clades, Clade I-V. Clade I, II and III included mainly Indian species whereas Clade V included mostly African species. Clade IV comprised D. serotinum. Clade I included nine taxa including our newly described species, D. mukaianum. The new species was phylogenetically placed with D. erythraeum, D. saxorum and D. ursulae. Morphologically, the species resembled D. montanum and D. ursulae but differed in characters such as tepal cohesion, number of ovules per locule and foul-smelling flowers. Clade II and III included 11 and six taxa, respectively. D. erythraeum which has a native range from Egypt to western India was found in Clades I and V. The widespread Dipcadi species, viz. D. erythraeum and D. serotinum showed polyphyly however, the monophyly of Dipcadi is established. Our studies suggest that additional molecular markers (plastid as well as nuclear) should be tested for their taxonomy utility. Further work on the historical biogeography of Dipcadi on the subfamily Ornithogaloideae with more genetic data will yield insights how aridification of the landscape would have shaped the evolution of the geographical clades.

Immune reconstitution inflammatory syndrome in a HIV-infected patient with disseminated tuberculosis.

Immune reconstitution inflammatory syndrome (IRIS) is the paradoxical worsening of pre-existing infectious processes after commencement of anti-retroviral therapy (ART) in HIV-infected patients. Its manifestations are dependent on the underlying opportunistic infections. We report a case of an HIV-infected patient with disseminated tuberculosis, who responded to anti-tuberculosis therapy but suffered from paradoxical worsening after commencement of ART.

Note: advancement in tip etching for preparation of tunable size scanning tunneling microscopy tips.

The two aspects of a scanning tunneling microscopy tip, the macroscopic profile and the nanoscale apex, can be tailored by controlling the tension during electrochemical etching and the solution-electrode contact area via acetone vapor. The apex diameter is shown to be proportional to the square root of the tension, and is demonstrated over apex diameters of 150-500 nm. The apex was found to be created in four distinct shapes where a secondary etching can reshape the tip into a single geometry. Improvement in tip height and stability of the profile are demonstrated versus a non-acetone fabrication control.

Clinical applications of platelet-rich plasma in patellar tendinopathy.

Platelet-rich plasma (PRP), a blood derivative with high concentrations of platelets, has been found to have high levels of autologous growth factors (GFs), such as transforming growth factor-ß (TGF-ß), platelet-derived growth factor (PDGF), fibroblastic growth factor (FGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). These GFs and other biological active proteins of PRP can promote tissue healing through the regulation of fibrosis and angiogenesis. Moreover, PRP is considered to be safe due to its autologous nature and long-term usage without any reported major complications. Therefore, PRP therapy could be an option in treating overused tendon damage such as chronic tendinopathy. Here, we present a systematic review highlighting the clinical effectiveness of PRP injection therapy in patellar tendinopathy, which is a major cause of athletes to retire from their respective careers.

Inhibition of mTOR affects protein stability of OGT.

Autophagy regulates cellular homeostasis through degradation of aged or damaged subcellular organelles and components. Interestingly, autophagy-deficient beta cells, for example Atg7-mutant mice, exhibited hypoinsulinemia and hyperglycemia. Also, autophagy response is diminished in heart of diabetic mice. These results implied that autophagy and diabetes are closely connected and affect each other. Although protein O-GlcNAcylation is up-regulated in hyperglycemia and diabetes, and O-GlcNAcylated proteins play an important role in metabolism and nutrient sensing, little is known whether autophagy affects O-GlcNAc modification and vice versa. In this study, we suppressed the action of mTOR by treatment of mTOR catalytic inhibitors (PP242 and Torin1) to induce autophagic flux. Results showed a decrease in global O-GlcNAcylation, which is due to decreased OGT protein and increased OGA protein. Interestingly, knockdown of ATG genes or blocking of lysosomal degradation enhanced protein stability of OGT. In addition, when proteasomal inhibitor was treated together with mTOR inhibitor, protein level of OGT almost recovered to control level. These data suggest that mTOR inhibition is a more efficient way to reduce protein level of OGT rather than that of CHX treatment. We also showed that not only proteasomal degradation regulated OGT stability but autophagic degradation also affected OGT stability in part. We concluded that mTOR signaling regulates protein O-GlcNAc modification through adjustment of OGT stability.

Protective effects of peroxiredoxin 6 overexpression on amyloid ß-induced apoptosis in PC12 cells.

Oxidative stress triggered by amyloid beta (Aß) accumulation contributes substantially to the pathogenesis of Alzheimer's disease (AD). In the present study, we examined the involvement of the antioxidant activity of peroxiredoxin 6 (Prdx 6) in protecting against Aß25-35-induced neurotoxicity in rat PC12 cells. Treatment of PC12 cells with Aß25-35 resulted in a dose- and time-dependent cytotoxicity that was associated with increased accumulation of intracellular reactive oxygen species (ROS) and mitochondria-mediated apoptotic cell death, including activation of Caspase 3 and 9, inactivation of poly ADP-ribosyl polymerse (PARP), and dysregulation of Bcl-2 and Bax. This apoptotic signaling machinery was markedly attenuated in PC12 cells that overexpress wild-type Prdx 6, but not in cells that overexpress the C47S catalytic mutant of Prdx 6. This indicates that the peroxidase activity of Prdx 6 protects PC12 cells from Aß25-35-induced neurotoxicity. The neuroprotective role of the antioxidant Prdx 6 suggests its therapeutic and/or prophylactic potential to slow the progression of AD and limit the extent of neuronal cell death caused by AD.

Adenosine A2A receptor polymorphisms in Korean patients with systemic sclerosis.

Adenosine A2A receptor (ADORA2A) regulates inflammation, promotes tissue repair and collagen production by human dermal fibroblasts. We investigated the genetic polymorphisms of ADORA2A in susceptibility to systemic sclerosis (SSc). We genotyped 142 Korean SSc patients and 150 controls for polymorphisms of -1751A/C (rs5996696) and 1976C/T (rs5751876), to cover the promoter and all exon sequences of ADORA2A in Koreans, using TaqMan fluorogenic 5' nuclease assay and single base primer extension assay. Neither -1751A/C nor 1976C/T polymorphism showed difference in the distribution of alleles or genotypes between patients and controls with allele frequency of 89.9% v 91.0% for -1751A (p=0.64) and 56.5% v 54.0% for 1976C (p=0.55). Our findings suggest that the role of ADORA2A in SSc may not be genetically related.

Whole body vibration and dynamic restraint.

The purpose of this study was to identify changes due to whole body vibration in peroneus longus (PL) activation following ankle inversion perturbation. Participants were 22 (age 22.1 +/- 1.8 yrs, ht 168.8 +/- 8.2 cm, mass 65.5 +/- 11.2 kg) physically active male and female students with no recent history of lower extremity injury. Measurements of PL electromechanical delay (EMD), reaction time, and muscle activation were collected from two groups (WBV and control) over 3 time intervals (pretreatment, posttreatment, and 30 min posttreatment). Two-way ANOVAs were used to compare groups over time for all dependent variables. No group x time interactions were detected (p < 0.05) for any of the dependent variables. Whole body vibration did not alter PL EMD, reaction time, peak EMG, or average EMG. The use of WBV for enhancing ankle dynamic stability was not supported by this study. However, more data are needed to determine if WBV is an effective intervention in other areas of injury prevention or rehabilitation. These data were not consistent with the hypothesis that WBV enhances muscle spindle sensitivity.

Molecular cloning and characterization of the copper/zinc and manganese superoxide dismutase genes from the human parasite Clonorchis sinensis.

A copper/zinc superoxide dismutase (Cu/ZnSOD) gene and a manganese superoxide dismutase (MnSOD) gene of the human parasite Clonorchis sinensis have been cloned and their gene products functionally characterized. Genes Cu/ZnSOD and MnSOD encode proteins of 16 kDa and 25.4 kDa, respectively. The deduced amino acid sequences of the two genes contained highly conserved residues required for activity and secondary structure formation of Cu/ZnSOD and MnSOD, respectively, and show up to 73.7% and 75.4% identities with their counterparts in other animals. The genomic DNA sequence analysis of Cu/ZnSOD gene revealed this as an intronless gene. Inhibitor studies with purified recombinant Cu/ ZnSOD and MnSOD, both of which were functionally expressed in Escherichia coli, confirmed that they are copper/zinc and manganese-containing SOD, respectively. Immunoblots showed that both C. sinensis Cu/ZnSOD and MnSOD should be antigenic for humans, and both, especially the C. sinensis MnSOD, exhibit extensive cross-reactions with sera of patients infected by other trematodes or cestodes. RT-PCR and SOD activity staining of parasite lysates indicate that there are no significant differences in mRNA level or SOD activity for both species of SOD, indicating cytosolic Cu/ZnSOD and MnSOD might play a comparatively important role in the C. sinensis antioxidant system.

Activity of phosphoforms and truncated versions of Ndt80, a checkpoint-regulated sporulation-specific transcription factor of Saccharomyces cerevisiae.

Ndt80 contributes to the highly regulated cascade of sequential gene expression that directs spore formation in Saccharomyces cerevisiae. This DNA-binding transcriptional activator, which is responsible for the expression of a set of middle sporulation-specific genes, is a target of the meiotic recombination checkpoint. Triggering of this checkpoint prevents phosphorylation and accumulation of active Ndt80. In this study we have investigated the requirements for the activation function of Ndt80 by exploring the role of phosphorylation in the regulation of its activity and by examining the effect of C-terminal truncations. Of three phosphoforms of Ndt80 that we resolved, which we refer to as P approximately Ndt80", P approximately Ndt80', and P approximately Ndt80 in order of increasing electrophoretic mobility, the P approximately Ndt80" and P approximately Ndt80' isoforms correlated with active Ndt80. In particular, P approximately Ndt80" was present in lysates from wild-type sporulating cells and in cells that bypassed checkpoint-mediated arrest as a result of mutations in RAD17, SUM1, or SWE1, or overexpression of NDT80. P approximately Ndt80' was the slowest-migrating isoform that accumulated in Delta ime2/Delta ime2 Delta sum1/Delta sum1 cells in sporulation medium and in mitotic cells that ectopically expressed NDT80. Nonphosphorylated Ndt80 and P approximately Ndt80, which had a slightly lower mobility than nonphosphorylated Ndt80 and was the predominant phosphoform present in checkpoint-arrested cells, correlated with inactive Ndt80. These data are consistent with the notion that extensive phosphorylation, but not Ime2-dependent phosphorylation, of Ndt80 is required for its activity. Examination of the effect of increasingly extensive truncation of the C terminal region of Ndt80 revealed that some functions of Ndt80 were more sensitive to a reduction in its activity than others. In particular, we found that a truncated version of Ndt80 that lacked the last 110 residues was able to promote expression of some middle sporulation-specific genes, but could not direct spore formation. Full activity, however, could be restored to this version of Ndt80 by increasing its level of expression.

Differences in embryonic development in sensitive and resistant matings to pregnancy block stimuli in mice.

Pregnancy block from exposure to foreign male mouse pheromones is sensitive to both male and female mating strain, as well as the foreign male pheromone-producing strain. Incidence of pregnancy block by male pheromones in mice is different depending on the combination of females, stud males and stimulus males. BALB/cA females mated with BALB/cA males showed a 100% pregnancy block when exposed to males of the DDK strain (Chung et al., 1997). In contrast, BALB/cA females mated with males of dissimilar strain show high rates of pregnancy even if they are exposed to DDK males; this difference is thought to be due to the difference in viability of embryos (Chung et al., 1999). The present study investigated how development of BALB/cA and F1 embryos differ under the influence of pregnancy block stimuli. F1 embryos had significantly higher numbers of cells than did the BALB/cA embryos (P<0.05) at day 3 of pregnancy after exposure to DDK males or after bromocriptine (dopamine agonist, 4 mg kg(-1), i.p.) treatment. Histological observation after bromocriptine treatment revealed that: (i) on day 4 of pregnancy, BALB/cA embryos tended to form a large blastocoel, but showed abnormalities such as degeneration of primitive endoderm and depression of the outer trophoblast-distal endoderm layer at the periphery of the inner cell mass (ICM) or detachment of the ICM from the outer layer. In contrast, 60-70% of F1 embryos were normal late blastocysts and incipient egg cylinders, but 28-40% of early blastocysts were degenerating; and (ii) day 5 BALB/cA embryos were in the range from incipient egg cylinder with a large proamniotic cavity to ectoplacental cone only, but their proximal endoderm and trophoblast-distal endoderm layer were degenerating. In contrast, the F1 embryos were mostly at the egg cylinder stage and maintained normal structure except for occasional enlargement of the developing yolk sac cavity. These results indicate that the lining of the inner surface of trophoblast by distal endoderm layer may be more firmly established and that the inner environment for development of F1 embryos may be more effectively maintained, thereby making them more resistant to deleterious influences due to pregnancy block stimuli than are BALB/cA embryos.

Safety of sputum induction in moderate-to-severe chronic obstructive pulmonary disease.

This retrospective study was designed to evaluate the safety and efficacy of a bronchoprotective sputum induction protocol in moderate to severe chronic obstructive pulmonary disease (COPD). Forty-two adults with COPD (FEV1 = 51.7 +/- 3.2% predicted (mean +/- SEM)) under went sputum induction using a protocol designed to minimize hypertonic saline-induced bronchoconstriction. Hypertonic (3%) saline was used for subjects with FEV1 > or = 50%, and normal (0.9%) saline was used for subjects with FEV1 < 50%. Primary outcomes were change in peak flow, FEV1 and oxygen saturation. Mean decline in peak flow during sputum induction was 13.2 +/- 2.1%. FEV1 fell by 11.4 +/- 2.3%, an absolute fall of 0.14 +/- 0.031. Oxygen saturation did not change. A fall in peak flow of > or = 20% reliably predicted a fall in FEV1 of > or = 20%. Thirty-five of 42 subjects (83.3%) produced an acceptable sputum sample. Sputum eosinophil and neutrophil percentages were 2.8 +/- 0.9 and 73.0 +/- 3.0%, respectively, and were not correlated with changes in peak flow, FEV1 or oxygen saturation. A protocol for sputum induction which restricts the use of hypertonic saline based on lung function is both safe and effective in subjects with moderate to severe COPD.

Distal lung dysfunction at night in nocturnal asthma.

We have previously shown that patients with nocturnal worsening of asthma (nocturnal asthma) exhibit increased parenchymal inflammation at night. To evaluate the functional significance of this parenchymal inflammation, 10 subjects with nocturnal asthma (NA), four subjects with non-nocturnal asthma (NNA), and four normal control subjects underwent bronchoscopy with measurement of peripheral airways resistance (Rp) at 4:00 P.M. and at 4:00 A.M. Employing a wedged bronchoscope technique, Rp was measured. Flow was stopped, and the pressure reached after 10 s of decay was termed the plateau pressure. The time constant of this decay (tau) was measured, and the peripheral compliance (Cp) was calculated as tau/Rp. The NA group exhibited the highest Rp values at 4:00 P.M. and at 4:00 A.M. as compared with the NNA and control groups, but all groups were significantly different from each other at 4:00 P.M.: NA, 0.113 +/- 0.02 cm H(2)O/ml/min; NNA, 0.033 +/- 0.005 cm H(2)O/ml/min; Control subjects, 0.010 +/- 0.001 cm H(2)O/ ml/min; p = 0.0001; and at 4:00 A.M.: NA, 0.129 +/- 0.023 cm H(2)O/ ml/min; NNA, 0.035 +/- 0.007 cm H(2)O/ml/min; Control subjects, 0.009 +/- 0.002 cm H(2)O/ml/min; p = 0.0003. None of the groups exhibited statistically significant differences in Rp between 4:00 P.M. and 4:00 A.M. The plateau pressure increased significantly from 4:00 P.M. to 4:00 A.M., but only in the NA group (7.7 +/- 0.9 cm H(2)O at 4:00 P.M. versus 16.9 +/- 4.6 cm H(2)O at 4:00 A.M.; p = 0.0004). Cp was decreased in the NA group as compared with the NNA and control groups at both 4:00 P.M. (p = 0.0003) and 4:00 A.M. (p = 0.003). The Rp positively correlated with the residual volume at both 4:00 P.M. (r = 0.71, p = 0.004) and 4:00 A.M. (r = 0.59, p = 0.03). We conclude that the distal lung units, specifically the collateral channels, and may be functionally altered at night in NA.

Role of Ha-ras activation in superficial papillary pathway of urothelial tumor formation.

Urothelial tumors develop along two distinctive phenotypic pathways (superficial papillary non-invasive tumors versus flat carcinoma in situ lesions), with markedly different biological behavior and prognosis. Although multiple genetic alterations have been identified in human bladder cancer, their cause-effect relationship with the two pathways has not been firmly established. Using a urothelium-specific promoter of the uroplakin II gene, we showed earlier in transgenic mice that the urothelial expression of SV40T antigen, which inactivates p53 and pRb, induced carcinoma in situ and invasive and metastatic bladder cancer. In striking contrast, we demonstrate here that the urothelial expression of an activated Ha-ras in transgenic mice caused urothelial hyperplasia and superficial papillary non-invasive bladder tumors. These results provide strong, direct experimental evidence that the two phenotypical pathways of bladder tumorigenesis are caused by distinctive genetic defects. Our results indicate that Ha-ras activation can induce urothelial proliferation in vivo; and that urothelial hyperplasia is a precursor of low-grade, superficial papillary bladder tumors. Our transgenic models provide unique opportunities to study the detailed molecular events underlying different types of bladder neoplasms, and can serve as useful preclinical models for evaluating the in vivo efficacy of preventive and therapeutic agents that act on various signaling pathways in bladder cancer.

Exhaled nitric oxide correlated with induced sputum findings in COPD.

STUDY OBJECTIVES: Neutrophilic airway inflammation may underlie the pathogenesis of COPD. We examined repeated measurements of the fractional concentration of exhaled nitric oxide (FENO) and the correlation with cells and mediators in induced sputum (IS) from patients with COPD. PARTICIPANTS: Eleven COPD subjects (9 men and 2 women, aged 46 to 69 years) with predicted FEV(1) of 45 to 70%. SETTING: A hospital research laboratory. DESIGN: Single-cohort, prospective study with four visits at two weekly intervals. INTERVENTIONS: FENO and spirometry were assessed at all visits, and IS for differential cell count, leukotriene-B(4) (LTB(4)) and interleukin (IL)-8, nitrite, and nitrate at visit 1, visit 3, and visit 4. RESULTS: During the study, there were significant declines in mean percent predicted FEV(1), from 55.2 to 51.6% (p = 0.029), and mean FEV(1)/FVC ratio, from 50.4 to 45.4% (p = 0.001), accompanied by a significant increase in FENO geometric mean (95% confidence limits), from 15.2 (10.9 to 21.2) to 23.6 (17.1 to 32.4) parts per billion (p = 0.037), and sputum LTB(4), from 1.79 (1.03 to 3.11) to 3.57 (1.95 to 6.53) ng/mL (p = 0.033), but no significant change in other sputum parameters. From visits 1 to 4, the change in percent neutrophils correlated with the changes in FENO and IL-8 (r = 0.648, p = 0.028; r = 0.60, p = 0.05, respectively). Hypertonic saline solution induction of sputum caused a fall in FEV(1), from 1.83 +/- 0.44 to 1.46 +/- 0.44 L (p = 0.049). CONCLUSIONS: The worsening spirometry results were accompanied by significant increases in FENO and sputum LTB(4). FENO may be related to neutrophilic inflammation driven by the chemoattractant IL-8. FENO and IS may be useful markers of airway inflammation in COPD patients. Sputum induction with hypertonic saline solution causes a significant fall in FEV(1) requiring appropriate caution.

Tamm-Horsfall protein binds to type 1 fimbriated Escherichia coli and prevents E. coli from binding to uroplakin Ia and Ib receptors.

The adherence of uropathogenic Escherichia coli to the urothelial surface, a critical first step in the pathogenesis of urinary tract infection (UTI), is controlled by three key elements: E. coli adhesins, host receptors, and host defense mechanisms. Although much has been learned about E. coli adhesins and their urothelial receptors, little is known about the role of host defense in the adherence process. Here we show that Tamm-Horsfall protein (THP) is the principal urinary protein that binds specifically to type 1 fimbriated E. coli, the main cause of UTI. The binding was highly specific and saturable and could be inhibited by d-mannose and abolished by endoglycosidase H treatment of THP, suggesting that the binding is mediated by the high-mannose moieties of THP. It is species-conserved, occurring in both human and mouse THPs. In addition, the binding to THP was much greater with an E. coli strain bearing a phenotypic variant of the type 1 fimbrial FimH adhesin characteristic of those prevalent in UTI isolates compared with the one prevalent in isolates from the large intestine of healthy individuals. Finally, a physiological concentration of THP completely abolished the binding of type 1 fimbriated E. coli to uroplakins Ia and Ib, two putative urothelial receptors for type 1 fimbriae. These results establish, on a functional level, that THP contains conserved high-mannose moieties capable of specific interaction with type 1 fimbriae and strongly suggest that this major urinary glycoprotein is a key urinary anti-adherence factor serving to prevent type 1 fimbriated E. coli from binding to the urothelial receptors.

Neurogranin null mutant mice display performance deficits on spatial learning tasks with anxiety related components.

Neurogranin/RC3 is a protein that binds calmodulin and serves as a substrate for protein kinase C. Neuronally distributed in the hippocampus and forebrain, neurogranin is highly expressed in dendritic spines of hippocampal pyramidal cells, implicating this protein in long-term potentiation and in learning and memory processes. Null mutation of the neurogranin gene Ng generated viable knockout mice for analysis of the behavioral phenotype resulting from the absence of neurogranin protein. Ng -/- mice were normal on measures of general health, neurological reflexes, sensory abilities, and motor functions, as compared to wild type littermate controls. On the Morris water task, Ng -/- mice failed to reach acquisition criterion on the hidden platform test and did not show selective search on the probe trial. In the Barnes circular maze, another test for spatial navigation learning, Ng -/- mice showed impairments on some components of transfer, but normal performance on time spent around the target hole. Abnormal and idiosyncratic behaviors were detected, that appeared to represent an anxiogenic phenotype in Ng -/- mice, as measured in the light<-->dark exploration test and the open field center time parameter. These findings of apparent deficits in spatial learning and anxiety-like tendencies in Ng -/- support a role for neurogranin in the hippocampally-mediated interaction between stress and performance.

A nicotinic cholinergic agonist (GTS-21) and eyeblink classical conditioning: acquisition, retention, and relearning in older rabbits.

Previously we demonstrated that GTS-21, a nicotinic cholinergic agonist, ameliorated eyeblink classical conditioning deficits in older rabbits. The present experiment was undertaken to replicate and extend these results by examining the effects of GTS-21 on retention and relearning. Retired breeder rabbits received 15 daily injections of 0.5 mg/kg GTS 21 (n = 8) or sterile saline vehicle (n = 8) during acquisition training, and no further injections occurred. Acquisition of conditioned responses (CRs) was significantly better in GTS-21-treated rabbits. During the first tone-alone retention session in week 6 of the experiment, rabbits initially treated with GTS-21 produced significantly more CRs than vehicle-treated rabbits. There were no group differences in retention at the 13-week retest. Differences in relearning were in the predicted direction but did not attain statistical significance. Results indicate that treatment with GTS-21 ameliorates learning beyond the period when the drug is actually administered.

The calpain small subunit gene is essential: its inactivation results in embryonic lethality.

Creation of transgenic (knockout) mice deficient in calpain small (30 kDa) subunit gene was undertaken to clarify the proposed role of the small subunit for calpain proteolytic activity and to gain insight into the importance of the gene in the whole animal. The gene was targeted and disrupted in embryonic stem cells by homologous recombination, and chimeric mice were generated. Heterozygous F1 generation mice were crossed to obtain F2 generation. Among F2 generation mice, we found only wild-type and heterozygous animals in the 80 pups genotyped to date; no homozygous mice have been found, although 20 were expected. The heterozygotes had no apparent phenotypic abnormalities. Analysis of their tissues revealed no significant difference in mRNA expression, protein content, or proteolytic activity in comparison with their wild-type littermates. Genotyping of fetuses at different stages of development also revealed only wild-type and normal heterozygous fetuses. No moribund embryos or resorption sites were observed in the uterine cavity. The results indicate that at least one normal allele is essential for postnatal survival. Disruption of both alleles appears to be lethal in very early fetal development.